May 10, 2010 - Here's the rub: using values of around 100 and 200 uL in the ... Anyway, the Test Plate set-up at this point is the first big/initial goal here, and ...
Example step-by-step PQ set-up ~
Example from 3-10-2011 jmg
Setting up PQ from scratch for a complex, multiplex bacterial gDNA qPCR set-up: 0.) Select the appropriate extinction coefficient for the nucleic acid you are working with by: From home position, activate the ~ C.H. Hidden VERSION rendered 5-10-2010/2-20-2011. Dedicated to my family…
Ctrl Ctrl x ISURF #03407 Shift F
Property of ISU-ISURF
To Quick Access
Ctrl m
To Printouts
Ctrl w
To LCM Adjust To Top
Simple Mix
SC
Reset
To Sample Aiming
Go To Questionnaire
LCM Add
MMRT
Primer File
TP Cq
button here:
To ERROR List
Inhib. Thresh
Quanta To MM Decoder
Probe? Back to 2008
Sample Dilutions
+Pb
To Point4 Select a
Free Decoder
Plates To Point Select b
Auto
And, since you are assaying dsDNA in your reactions, click the small red button in either the left portion of cell S153, or the left portion of cell U128, to tell the PQ program that you will be using the extinction coefficient for dsDNA when the program interprets your entered A260nm readings for your samples: Extinction coefficients to use in cell F15 If initial template is ssRNA: 40 If initial template is ssDNA: 37 If initial template is oligo ssDNA: 33
g/mL/1 o.d. @ 260nm
If initial template is dsDNA: 50
g/mL/1 o.d. @ 260nm
g/mL/1 o.d. @ 260nm g/mL/1 o.d. @ 260nm
/1000 0.04 0.037 0.033 0.05
Select coefficient
x
Ctrl Shift Z
or
ssRNA ssDNA oligo dsDNA
Quick-calc RNAor DNA od260nm
ng/uL
Calc.'d 260nm
50 50 1
dilution? 1: 1 (use in P137?)
1.) Enter primer-probe names and desired final qPCR rxn primer-probe [nM] into UMES.xls range F126:J132, and enter “t” or “h” into range K126:132 to designate each as “target” or as “housekeeper” (reference gene). Enter “t” for all here, as DNA contamination is not a concern. (Note, at anytime you can get to home position by clicking on a star wherever you see it…) Ctrl m
~ C.H. Hidden VERSION rendered 5-10-2010/2-20-2011. Dedicated to my family…
Ctrl Ctrl x ISURF #03407 Shift F
Property of ISU-ISURF
TP Cq
Reset Auto fill 7 like 1st
To Quick Access
To Printouts
Ctrl w LCM Add
MMRT
To LCM Adjust To Top
Simple Mix
Primer File
SC To Sample Aiming
Go To Questionnaire
To ERROR List
Inhib. Thresh
Quanta To MM Decoder
+Pb
Free Decoder
Probe? Back to 2008
Sample Dilutions To Point4 Select a
Ctrl Shift D Invitrogen MM+Pb param.
Plates To Point Select b
ENTER TARGET AND FINAL IN-WELL [nM] PRIMER-PROBE INFO Fix Species Target Name Fwd [nM] Rev [nM] Probe [nM] eae 250 250 bact Z5214 250 250 bact HKG 200 200 bact Z5211 250 250 bact
All stock primer/prob
Name: Largest summed nM in top row
The anom
For PREXCEL-Q default Two-Step qPCR use only
Select up to 7 genes o Enter "t" for Target an for Housekeeper (Nor or Endogenous refere t t t t
2.) Enter “10” into cell M117 to tell the system what your initial Stock uM primer and probe All stock primer/probe concentrations: 10 M concentrations are:
3.) Enter sample information into UMES.xls cell range A120:C154
4.) Then activate two buttons: First, the ALL x's button near cells F117:118, and then the P aste to abo ve button near cells B118:119 IMPORTANT! Then adjust cell F2 to “20” (each sample contributes this many uL to Stock I). Calculate this manually from Plate drawings. (Generally 200 to 800 uL of total Stock I volume is sufficient for most situations: having extra on hand is good.
This particular section (below), would be filled out as follows if each 90-uL sample were to be used for PQ (and initially pre-diluted 1:5, etc.) for qPCR purposes: 5.) In range F134:P139, enter all your parameters for sample size, RNase treatment, qPCR reaction parameters, place an “x” in cell H137 and J139, and enter the blanket qPCR pre-dilution of samples (1:5), and the fact that samples were not diluted for NanoDrop readings (1:1) (not 1:0). Questionnaire Minimum initial DNA sample isolate volume: 100 uL Ctrl Shift F (enter 1 or 2 here for NanoDrop) L 2 uL Total volume prepared for Spec. or NanoDrop 2 uL DNA actual or theoretic part of that volume U/uL RNase: 1 Already Rxn vol. total OFF: enter sample vol. into K138 only x ~0.1646 90 uL DNase file XNased Sample treated 90 uL RNase rxno size Enter post-XNased sample volume here and into K138 90 uL Min allowed 260nm: make K139 = K138 "x" ~0.1646 x 90.00 uL Recovered 0 uL Stop sol'n. volume
20.00 uL DNA added/rxn qPCR 3.00 uL performed Max Cust Used in Wells GIT-based 20.00 uL (or Trizol) One-Step
ng/uL RNA 20 uL ea. cDNA made: 200 uL RT rxn size
Two-Step RT:
uL cDNA adjust: 0 uL
Spec./Nano @ 1: 1 Prediluted 1: 5
directly if needed - although it is not in red font)
BUT …
A260nm dilution (samples before use)
OFF:
Ignore these inputs
BUT, since we initially decided to use only 35 uL of the original (90-uL) samples, filling out this particular section is more accurately performed as follows: (which allows PQ to tell you if 35 uL of each sample would be enough to start with to perform everything for the entire qPCR) 5.) In range F134:P139, enter all your parameters for sample size, RNase treatment, qPCR reaction parameters, place an “x” in cell H137 and J139, and enter the blanket qPCR pre-dilution of samples (1:5), and the fact that samples were not diluted for NanoDrop readings (1:1) (not 1:0). Questionnaire Minimum initial DNA sample isolate volume: 100 uL Ctrl Shift F (enter 1 or 2 here for NanoDrop) L 2 uL Total volume prepared for Spec. or NanoDrop 2 uL DNA actual or theoretic part of that volume U/uL RNase: 1 Already Rxn vol. total OFF: enter sample vol. into K138 only x ~0.1646 35 uL DNase file XNased Sample treated 35 uL 35 uL RNase rxno size Enter post-XNased sample volume here and into K138 Min allowed 260nm: make K139 = K138 "x" ~0.1646 x 35.00 uL Recovered 0 uL Stop sol'n. volume
20.00 uL DNA added/rxn qPCR 3.00 uL performed Max Cust Used in Wells GIT-based 20.00 uL (or Trizol) One-Step
ng/uL RNA 20 uL ea. cDNA made: 200 uL RT rxn size
Two-Step RT:
uL cDNA adjust: 0 uL
Spec./Nano @ 1: 1 Prediluted 1: 5
directly if needed - although it is not in red font) S im ple
A260nm dilution (samples before use)
OFF:
6.) Activate the button in the middle of the “X” near cell H118 in the large M ix “PREXCEL-Q” logo. This tells the system that you are using a simple 2X qPCR mix for simple one-step DNA template qPCR.
Ignore these inputs
7.) Activate the Fix button near the right edge of UMES.xls cell J124 (this tells the system your target names and target primer-probe concentrations)… 8.) Fill in the appropriate cells in the J1:O28 region to tell the system your Test Plate and Final Plate parameters (as well as number of points to be prepared (from Stock I) for your final standard curves): TEST PLATE PARAMETER ENTRY AREA
Minimum of each sample needed:
ng/uL
50 uL
7.07 uL Clear J1
1 ample & NRC Plates
Target 1
eae
1
prepared for Test
Z5214
Target 2
Z5214
1
Plate purposes
HKG
Target 3
HKG
1
adjust
Z5211
Target 4
Z5211
problem samples
1.625
Target 5
50.00 uL
Target 6
1 1 1
Target 7
1
15 uL Safe amount of each sample for Test Plate; when each sample is used in singlet
RNA isolation (Trizol, Column, LCM)
Trizol samples 90
SC
To Sample Aim
Test Plate
xr,r,mr,m,n,a,sa
MMRT
33.3333
4
Plates
93.45248856
110 uL ~415 uL 0.23521 ined optimal
LIMITING SAMPLE
This is the list of all the targets chosen to be tested in 50 this study. A maximum of 14 targets can be tested 4 at a time.
Target abundance
In-well Dilutions used 1:
To Extra Stock I area
Both Ctrl y and Ctrl Shift Z
1
35 1 5 1 0 40 40 0
iterations
eae
To Printouts
0
Targets Tested
Sample sizes
557 uL
Not enough RNA in
qPCR Target List
262.0010285 734.5394436 2059.336169 5773.502692 16186.44583 45379.90931 127225.9637 356687.4874 1000000 Auto-Attenuate
2
Inhib. Thresh
100000
use in M28
adjust:
Ctrl Shift Z
1.21 Extra MM
To MM Decoder
adjust 5.5
Ctrl Shift A
T 4
f.s. actually needed: 0 uL To Home
MMRT
Ctrl y
m
TP Cq
1.21
can Manually Adjust this (For Ctrl Shift A) Desired Upper Dilution
1: 1000000
Activate Ctrl Shift K to Reset PREXCEL-Q to original state Ctrl Shift F for original user targets & o.d.s
15.29 OK OK OK GOOD OK To Printouts
Sample reps: Wells per target: Adjust # of wells:
MM adjust Adjust this value to make sure you have enough Master Mix ("1" for quick yes-no set-ups)
1 12 -4
Ctrl Shift A Put large
So, to break down step 8 (above) into its particular, inductive maneuvers: (These particular maneuvers are all about planning ahead, using a “future-based mind-set/attitude” at this point …)
a.) Since bacterial DNA, overall, is very robust, running the 11 Test Plate Stock I dilutions from fullstrength out to a 1:1,000,000 dilution is OK, so, enter 1,000,000 into cell M28 and activate the button… (even though you may be looking for one copy of something in the end). C t rl S hif t A b.) Since your sample size is 3 uL per reaction, and you are multiplexing, preparing 50 uL of each dilution of Stock I for Test Plate purposes is plentiful, safe over-kill amount (and easier to prepare, ergonomically, than smaller amounts) – so, enter “50” into cell K2 for Test Plate sample volumes made. c.) In the cell region J17:J21 (blown up below), enter “4” into J17 and “1” into J20 to tell the system you will be using 5-point standard curves for each target on the Final Plates. Leave the default value “1” in cell J18 and “0” in J21, but, since you have 5 different control reactions you’d like to run on your Final Plates (NTCWater, NTCMOPS/EDTA, NAC1, NAC2 and NAC3), enter “5” into cell J19: Sample Plates (wells per target):
90
# of Samples:
35
MMRT
# Points in Standard Curves:
4
Plates
adj.# Smpls
0
Repeats of Standard Curves: NTCs used per target: e Extra Standard (1 or 0): es Fine Sample Adjust: nal NRC Plates (wells per target): or genomic DNA (NRC, NAC or NPC): Q-M # of NTC wells tested:
SC
1 5 1 0 40 40 0
The acronym “NTC” is thus loosely used here to connote all assay control-type reactions… To E Stoc
d.) Since we are only running an NTC on the Test Plate (in addition to the 11 dilutions of Stock I), we now need to adjust the # of samples explored on the Test Plate by placing a “-4” into cell O27 to tell the Test Plate function that it will only be running 12 samples total (1 NTC + 11 dilutions of Stock I = 12). Or else the program might think that your 4 other assay controls are being Tested too. So put Inhib. a -4 here:
ndance
n,a,sa
use in M28
adjust: 1.21
Z
Extra MM M er
adjust 5.5
ift A
T 4
To Home
MMRT
Ctrl y 00
f.s. actually needed: 0 uL
Thresh
1.21
ally Adjust this Shift A) pper Dilution
1: 1000000
Activate Ctrl Shift K to Reset PREXCEL-Q to original state Ctrl Shift F for original user targets & o.d.s
15.29 OK OK OK GOOD OK To Printouts
Sample reps: Wells per target: Adjust # of wells:
MM adjust Adjust this value to make sure you have enough Master Mix ("1" for quick yes-no set-ups)
1 12 -4
Ctrl Shift A Put large
e.) Realize that the default setting for PQ is to run duplicate wells (for standards, samples and any controls) on the Final Plates, but only singlet wells on the Test Plate. These adjustments (for technical replicates), if desired to be other than the default settings, are adjusted directly in cell O25 for the Test Plate, and U25 for the Final Plates. But, realize, if using other than a “1” technical replicate setting for the Test Plate, the graphic (depiction) that is printable from the PQ program of the Test Plate will not be correct. It defaults to only showing you singlet wells for that. All other
parameters regarding Mastermix will be correct however. Now, it is important at this point to draw out the desired layouts of your Final Plates so your are intimately familiar with how much of each sample and standard will need to be prepared as you go along toward your final set-up(s). So, to T o P rint o ut s begin this important part of the process, active the button (where-ever you may see it anywhere in the program), e.g. here, and go to the “Plates” tab (worksheet) inside that file ndance
n,a,sa
Inhib. Thresh
use in M28
adjust: 1.21
Z
Extra MM M er
adjust 5.5
ift A
T 4
To Home
MMRT
Ctrl y 00
f.s. actually needed: 0 uL
1.21
ally Adjust this Shift A) pper Dilution
1: 1000000
Activate Ctrl Shift K to Reset PREXCEL-Q to original state Ctrl Shift F for original user targets & o.d.s
15.29 OK OK OK GOOD OK To Printouts
Sample reps: Wells per target: Adjust # of wells:
MM adjust Adjust this value to make sure you have enough Master Mix ("1" for quick yes-no set-ups)
1 12 -4
Ctrl Shift A Put large
wherein cell range H3:BI137 depicts adjustable plate scenarios that you can mold and tweak to your specifications for your Final Plate layouts (see next page):
f.) Draw out your Final Plate layout(s) here to get a visual from which you can mentally calculate sample and standard volumes needed to complete your entire qPCR for all samples and standards for all targets. g.) Then, once you have those values in mind, catch the nearest star back to home position and activate the navigational button To Quick (above the “E” in the big PREXCEL-Q logo) to go to the Access
sample and standard volume entry cells F55 and F58, respectively, and enter your needs there: The internal reasoning for your scenario (although the program automatically calculates this too – but we’ll forego that for the moment to keep the “whys” of the maneuvers clear)… the internal reasoning for your needs would go as follows: for Final Plates, you have 4 targets, and 4 standard curves, each tested in duplicate, but in multiplex fashion. So, a raw 6 uL of each appropriately-diluted sample (dilutions not yet known since the Test Plate has not been run at this point) is needed to accomplish your Final Plates. Since 35 uL of each original sample was inflated by 1:5 dilutions… you already have ample sample material to create Stock I from and also more than enough to dilute further on an individual basis to attain the “red dot” dilution (thinking futuristically) upcoming…
Thus, to make things even easier, we’ll prepare 200 uL of each sample dilution and 100 uL of each final standard dilution. We do this by entering as follows into F55 and F58: Master Volume Control Setting 1 Choose Sample Amounts: 50 uL of each DNA U Master Volume Control Setting 2 Choose Standard Amounts: C 100 uL of each standard
This “200 uL” is the final volume of each normalized sample as shown on p. 33 of this document …
The “50” value here internally becomes 200 uL in the program, since 50 uL x 4 targets = 200 uL. E.g. if we assume we will be working within the same dynamic range for standard curves and sample dilutions for all targets (which is an ergonomically-sound initial assumption to always make), then, the program will assume to take the number of targets times the F55 value of “50” here to get “200.”
These values are very safe “over-kill” volumes for both samples and standards since the raw needs of each is really only 6 uL (appropriately-diluted) per sample, and 6 uL of each appropriately-diluted standard. (This of course is all due to the economy of sample- and standard-usage that multiplexing ultimately grants its fortunate user). Here’s the rub: using values of around 100 and 200 uL in the end for these things makes the pipettable volumes much easier to work with (since working with fractions of a uL is difficult and not advised anyway, scientifically nor ergonomically). In addition, having extra amounts of these things on hand (safe extra amounts) – without impinging on the amounts left over of your very original sample isolates, is also a very good thing in the event that Test Plates and or Final Plates have to be re-run for some unforeseen reason, and/or if you might introduce additional targets into more Test Plates and/or Final Plates for this particular sample set… (The “PQ diatribe” gets fairly thick here, I realize – so I must offer my apologies for the long-winded nature of all of this. I often pity the receivers of this program at this point – I really do; even though it all has worked perfectly every time. It is still a behemoth of sorts). But, the underlying premise of the monster is yet very simple: It uses the samples themselves to get a preliminary measure of each target amplification efficacy by virtue of the “Stock I/Test Plate/then Final Plates using Stock I-rendered standard curves work-flow idea.”
Anyway, the Test Plate set-up at this point is the first big/initial goal here, and that is what every maneuver so far has been leading up to (even though it may not seem evident just yet) … so: 9.) At this point, activate the button
C t rl Sh Z
C t rl S hif t Z
(these are both the same) to incorporate all of
the parameters you have suggested/entered into the program thus far. (Takes about a minute or so for this Macro to do its thing here). This particular button is found ubiquitously throughout the program so it can be activated whenever and wherever the user thinks the program needs to be informed and updated as to the deeds the user has perpetrated on the program at any point. (The program needs to be aware of what has been done to it; what values have been changed or added etc.)…
10.) Once the Macro has finished, it is time to finalize the regions of the program that are needed as printouts for the Test Plate: T o P rint o ut s First activate the button and go to the “TP Diltns” tab (worksheet) within that file. Then, use the navigational button T o Equal to move over to range S1:AA12 where you will see your Vo lumes (now familiar) Test Plate Stock I dilutions for your Test Plate:
1: 33.33 (uL) COMPREHESIVE SERIAL DILUTION TABLE (uL)
(Stock I) A Total made B 77.7 uL C 77.7 uL D 77.7 uL E 77.7 uL F 77.7 uL G 77.7 uL H 77.7 uL I 77.7 uL J 77.7 uL K 77.7 uL
Achieved
Actual Final Dilutions Achieved for Test Plate after used in-well:
Stock I
Water
to Next
(FINAL VOL.)
Dilutions 1:
27.7 uL
50.0 uL
27.7 uL
50.0 uL
2.8
27.7 uL
50.0 uL
27.7 uL
50.0 uL
7.9
27.7 uL
50.0 uL
27.7 uL
50.0 uL
22.0
27.7 uL
50.0 uL
27.7 uL
50.0 uL
61.8
27.7 uL
50.0 uL
27.7 uL
50.0 uL
173.2
27.7 uL
50.0 uL
27.7 uL
50.0 uL
485.6
27.7 uL
50.0 uL
27.7 uL
50.0 uL
1361.4
27.7 uL
50.0 uL
27.7 uL
50.0 uL
3816.8
27.7 uL
50.0 uL
27.7 uL
50.0 uL
10700.6
27.7 uL
50.0 uL
0.0 uL
77.7 uL
30000.0
1: 1: 1: 1: 1: 1: 1: 1: 1: 1:
93.45249 262.001 734.5394 2059.336 5773.503 16186.45 45379.91 127226 356687.5 1000000
The factors to tell the machine the relative strengths of these dilutions are also shown here (albeit vertically) in cells AA13:23 1 0.356687487 0.127225964 0.045379909 0.016186446 0.005773503 0.002059336 0.000734539 0.000262001 9.34525E-05 3.33333E-05
Print these things out…
11.) Now, go to the “TestPlate” tab (worksheet) within this same file either by tabbing directly T o P rint o ut s to it using the “TestPlate” tab at he bottom of the file or, again by using the navigational button (in cell Y15) here (which always takes you to the “TestPlate” tab within this file) … and you should see the depiction of your Test Plate (albeit not in Multiplex format): TEST PLATE DEPICTION: Machine Factors:
0.03
Scientific notation:
1.487
1.487
0.01070062 0.0038168
1.487
1.487
1.487
0.0013614 0.00048559 0.0001732
1.487
1.487
1.487
1.487
1.487
6.178E-05
2.204E-05
7.86E-06
2.8036E-06
0.000001
3.00E-02
1.07E-02
3.82E-03
1.36E-03
4.86E-04
1.73E-04
6.18E-05
2.20E-05
7.86E-06
2.80E-06
1.00E-06
2
3
4
5
6
7
8
9
10
11
12
1
Ideal DCts
A
NTC
1:
33.33
1:
93.5
1:
262.0
1:
734.5
1:
2059.3
1:
5774
1:
16186
1:
45380
1:
127226
1:
356687
1:
1000000
ALL 4
B
NTC
1:
33.33
1:
93.5
1:
262.0
1:
734.5
1:
2059.3
1:
5774
1:
16186
1:
45380
1:
127226
1:
356687
1:
1000000
Z5214
C
NTC
1:
33.33
1:
93.5
1:
262.0
1:
734.5
1:
2059.3
1:
5774
1:
16186
1:
45380
1:
127226
1:
356687
1:
1000000
HKG
D
NTC
1:
33.33
1:
93.5
1:
262.0
1:
734.5
1:
2059.3
1:
5774
1:
16186
1:
45380
1:
127226
1:
356687
1:
1000000
Z5211
E
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
F
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
G
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
28.2252
Relative factors ng DNA/well:
Relative factors
10.0675757 3.5909783 1.28085702 0.45686567 0.1629583 0.05812518 0.0207325 0.00739503 0.00263772
Tested concentrations to see where inhibition of any kind lets up for each different target … 1 0.35668749 0.127226 0.04537991 0.01618645 0.0057735 0.00205934 0.0007345 0.000262 condensed: Given that our Stock I Solution DNA mixture is calculated to be: 9.4084 ng/uL (made up of 1: 5 - diluted
0.00094084
9.3452E-05 3.33333E-05 DNAs: post-RNAse Treatment)
12.) Alter the picture manually to agree with what your multiplex Test Plate set-up actually is (simply delete the areas that do not apply and rename the 1st target to “ALL” or “ALL 4” etc.) and print out the desired region of this file for an in-lab copy of what the set-up looks like: TEST PLATE DEPICTION: Machine Factors: Scientific notation:
0.03
NTC
1.487
1.487
1.487
1.487
0.0013614 0.00048559 0.0001732
1.487
1.487
1.487
1.487
1.487
6.178E-05
2.204E-05
7.86E-06
2.8036E-06
0.000001
3.00E-02
1.07E-02
3.82E-03
1.36E-03
4.86E-04
1.73E-04
6.18E-05
2.20E-05
7.86E-06
2.80E-06
1.00E-06
2
3
4
5
6
7
8
9
10
11
12
1 A
1.487
0.01070062 0.0038168
1:
33.33
1:
93.5
1:
262.0
1:
734.5
1:
2059.3
1:
5774
1:
16186
1:
45380
1:
127226
1:
356687
1:
B
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
C
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
D
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
E
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
F
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
G
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
1:
Relative factors ng DNA/well:
Relative factors
28.2252
10.0675757 3.5909783 1.28085702 0.45686567 0.1629583 0.05812518 0.0207325 0.00739503 0.00263772
Tested concentrations to see where inhibition of any kind lets up for each different target … 1 0.35668749 0.127226 0.04537991 0.01618645 0.0057735 0.00205934 0.0007345 0.000262 condensed: 5 - diluted Given that our Stock I Solution DNA mixture is calculated to be: 9.4084 ng/uL (made up of 1:
1000000
Ideal DCts
ALL 4
0.00094084
9.3452E-05 3.33333E-05 DNAs: post-RNAse Treatment)
13.) Mastermix set-up acquisition: First catch the nearest star
Ctrl m
~ C.H. Hidden VERSION rendered 5-10-2010/2-20-2011. Dedicated to my family…
Ctrl Ctrl x ISURF #03407 Shift F
Property of ISU-ISURF
TP Cq
Reset Auto fill 7 like 1st
To M M Deco der
to home position, and, once there, activate the To Quick Access
To Printouts
Ctrl w
MMRT
To LCM Adjust To Top
Simple Mix
Primer File
SC To Sample Aiming
Go To Questionnaire
LCM Add
To ERROR List
Inhib. Thresh
Quanta To MM Decoder
+Pb
Free Decoder
Probe? Back to 2008
Sample Dilutions To Point4 Select a
Plates To Point Select b
ENTER TARGET AND FINAL IN-WELL [nM] PRIMER-PROBE INFO Fix Species Target Name Fwd [nM] Rev [nM] Probe [nM] eae 250 250 bact Z5214 250 250 bact HKG 200 200 bact Z5211 250 250 bact
button
And, once at the destination, it should look something like the following: GOOD
FIX
FIX2
20.00 uL prepared/Well 20.00 uL used per Well Depends on Machine/MM used
SYBR?:
Target Master Mix Set-up(s) 480.00 uL 0.00 uL 288.00 uL 768.00 uL
2X Master Mix: 0X RT-Taq Solution: Water: Total MMRT prepared:
1
Total MMRT needed:
eae Fwd primer:
6.00 uL
split
Rev primer:
0.00 uL
into
Probe:
6.00 uL
12
MMRT: 192.00 uL 0.00 uL water: 3
0.00 uL Copy to Free MM Decoder
To Free MM Decoder
then add
Extra/Target
Attain xtra
768.00 uL
2
0.00 uL
Z5214
xtra made
Less
X4
Reduce Xtra
X4 Less
Sample Plate(s)
To Printouts
0.00 uL
RNA added/well:
250 nM FWD primer 0 nM REV primer
Desired xtra samples prepared
0
93.5 uL
Master adjust: T split
Rev primer:
0.00 uL
into
Probe:
6.00 uL
MMRT: 192.00 uL water: 4
wells prepared
3.00 uL 48 48
1
1
Round
1 1.45833333
6.00 uL
0.00 uL Main Primer-Probe nM
Well size prepared: 20.00 uL
Fwd primer:
3.00 uL Sample to each
xtra ea.made
Total MM ea. 204.00 uL
More
More
17.00 uL amounts
Attain ALL xtras
0.00 uL
12
Test Plate Final Plates
17.00 uL amounts then add
NO EXTRA
3.00 uL Sample to each
To NRC
Enter up to 44 targets
5/10/2010
1 2 3
eae Z5214 HKG
4 5 6 7 8 9 10 11 12 13 14
Z5211
NRC target
To NRC -375 suggested xtra entry Reduce
The white font “T” here – is telling you that the program knows you are in “Test Plate” mode. So it knows that mastermix calculations at this point are for the Test Plate, and not for the Final Plates. When we approach the Final Plate set-ups (later on in this manifesto), we merely activate the Final Plates button here to send PQ off on its fetch mission to find the Final Plate mastermix parameters. Copy to Free MM Decoder
14.) Activate the button. This copies all things from here over to the “Free Mastermix decoder” (“qPCRUniversalPQMixDecoder12-1-09.xls”) file (which has universal flexibility to accommodate pretty much any possible master mix on the planet). And it is here that we first inform the PQ system that we are performing a Multiplex qPCR fantastica! (as opposed to a boring, drawn-out Monoplex soap opera, like I usually do…)
We inform PQ of the upcoming Multiplex approach by: 15.) Altering the Target entry area to tell it, effectively, that we are running only 1 target (although we know it is 4 targets at the same time). We do this by entering only one target name into the region in cells L2:L5 that now look like this: Enter up to 44 targets
5/10/2010
1 2 3
eae Z5214 HKG
4 5 6 7
Z5211
NRC target
But we now alter it to look like this: Enter up to 44 targets
5/10/2010
1 2 3 4 5 6 7
ALL4
NRC target
16.) Then we enter your desired multiplex primer-probe usages inside the “StrengthFactors” tab (sub-worksheet) inside this file:
Go ahead and activate this tab to go to this sub-worksheet…
Inside this sub-worksheet, your particular entries would look like: (adjust values in red only) Multiplexing: Number of targets? 4 Desired final "strength factor" Final sought mixture volume 5.000000000000000000X nM 500 uL 526.31579 uL Stock nM Use 10000 250 FWD: 62.50 uL Target 1 10000 0 REV: 0.00 uL 10000 250 Probe: 62.50 uL 10000 10000 10000
250 0 250
FWD: 62.50 uL Target 2 REV: 0.00 uL Probe: 62.50 uL
10000 10000 10000
200 0 200
FWD: 50.00 uL Target 3 REV: 0.00 uL Probe: 50.00 uL
10000 10000 10000
250 0 250
No of oligos:
8 1900
FWD: REV: Probe: Water: Total mix: collective nanomolarity
62.50 uL Target 4 0.00 uL Eliminate 62.50 uL water 25 uL 500 uL
Once you have entered the pertinent values in red, activate the “Use” button. TOP Then activate the button there (which should be somewhere in your field of view) and which should bring you to the following scene (see the next page):
GOOD
FIX
Target Master Mix Set-up(s)
20.00 uL used per Well Depends on Machine/MM used
SYBR?:
Total MMRT prepared:
1
oligo mix used
ALL4
48.00 uL
6 uL ea. of 8 oligos
48.00 uL
split
ignore
0.00 uL
into
ignore
0.00 uL
12
MMRT:
156.00 uL 0.00 uL
Extra/Target
20.00 uL
RNA added/well:
3.00 uL
-20
Desired xtra samples prepared
12
wells prepared
12
93.5 uL
Attain xtra
Master adjust: 2
xtra made
1/2X
1 Round
17.00 uL amounts
Fwd primer:
1
1.45833333 1
Probe:
Sample Plate(s)
MMRT:
5X oligo mix used
water:
Custom xtra
3.00 uL Sample to each
1 NO EXTRA
Rev primer:
RESET
0.00 uL
0.00 uL
Well size prepared:
0.00 uL
2X
then add
xtra ea.made
Total MM ea. 204.00 uL
156.00 uL
Total MMRT needed:
ignore
water:
120.00 uL 0.00 uL 36.00 uL 156.00 uL
2X Master Mix: 0X RT-Taq Solution: Water:
20.00 uL prepared/Well
Attain ALL xtras
Notice here I have covered up some values with a white rectangle here – so they are not redundantly considered as part of your mastermix set-up accidentally … But, you will see that the word “ignore” is beside the cells to not use anyway … so, I just put the blank box over these for now... Now I will remove the blank box: GOOD
FIX
Target Master Mix Set-up(s)
20.00 uL used per Well Depends on Machine/MM used
SYBR?:
Total MMRT prepared:
1
oligo mix used
ALL4
48.00 uL
6 uL ea. of 8 oligos
48.00 uL
split
ignore
0.00 uL
into
ignore
0.00 uL
12
MMRT:
156.00 uL 0.00 uL 0.00 uL
xtra made
Attain xtra
0.00 uL
Well size prepared:
20.00 uL
RNA added/well:
3.00 uL
-20
Desired xtra samples prepared
12
wells prepared
12
93.5 uL
Master adjust: 2 1/2X
RESET
Fwd primer:
1 Round
1 NO EXTRA 1
1.45833333 1
Rev primer: Probe:
Sample Plate(s)
MMRT:
5X oligo mix used
water:
3.00 uL Sample to each
xtra ea.made
Total MM ea. 204.00 uL
0.00 uL
2X
17.00 uL amounts then add
Extra/Target
156.00 uL
Total MMRT needed:
ignore
water:
120.00 uL 0.00 uL 36.00 uL 156.00 uL
2X Master Mix: 0X RT-Taq Solution: Water:
20.00 uL prepared/Well
Attain ALL xtras
Custom xtra
17.) To calculate extra amounts made (safe extra so you don’t run out of Mastermix during setting the reactions up physically in the lab), one adjust cells I5 and O13 in this file and then activates the button: to attain the desired parameters. One should work with these settings awhile Attain xtra until satisfactory values for safe extra amounts made are obtained… You will also see that you can type the desired extra amounts directly into cell H4 for extra amount left over after filling all tubes with primers-probes/mastermix, and cell O12 for how much extra initial master mix you have before adding the primers-probes to it. These values are exactly attained by again activating the Attain xtra button. And, to round up the primer-probe value to the nearest whole integer (for ergonomic, pipetting-related reasons), you can activate the Round button to attain that. There are other convenient functionalities here to tweak to infinity … but you can easily discover them on your own (the buttons are named somewhat intuitively here and there)… hopefully…
The settings used for your Test Plate would have ideally been (the Test Plate you ran, I set up the parameters before you told me that one of the targets like 200 nM primer and 200 nM probe): GOOD
FIX
Target Master Mix Set-up(s)
Depends on Machine/MM used
SYBR?:
Total MMRT prepared:
1
oligo mix used
ALL4
60.00 uL
7.5 uL ea. of 8 oligos
60.00 uL
split
ignore
0.00 uL
into
ignore
0.00 uL
12
MMRT:
195.00 uL 0.00 uL 0.00 uL
51.00 uL
Attain xtra
xtra made
1/2X
RESET
Fwd primer:
20.00 uL
RNA added/well:
3.00 uL
-20
Desired xtra samples prepared
12
wells prepared
12
60 uL
1.25 Round
1 NO EXTRA 1.25
1.03529412 1
Rev primer: Probe:
Sample Plate(s)
MMRT:
5X oligo mix used
water:
3.00 uL Sample to each
Well size prepared:
Master adjust: 2
20.68 uL
2X
17.00 uL amounts then add
Extra/Target
xtra ea.made
Total MM ea. 255.00 uL
195.00 uL
Total MMRT needed:
ignore
water:
165.91 uL 0.00 uL 49.77 uL 215.68 uL
2X Master Mix: 0X RT-Taq Solution: Water:
20.00 uL prepared/Well 20.00 uL used per Well
Attain ALL xtras
Cell O12 is set to “1.2” here (not seen in the field of view above) …
Custom xtra
Once you have your Test Plate Cq values, you: 18.) Enter them into UMES.xls file cell range C236:C246 by first catching the nearest star to home position and then activating the TP button here Cq Ctrl m
~ C.H. Hidden VERSION rendered 5-10-2010/2-20-2011. Dedicated to my family…
Ctrl Ctrl x ISURF #03407 Shift F
Property of ISU-ISURF
TP Cq
Reset
To Quick Access
To Printouts
Ctrl w LCM Add
MMRT
To LCM Adjust To Top
Simple Mix
Primer File
SC To Sample Aiming
Go To Questionnaire
To ERROR List
Inhib. Thresh
Quanta To MM Decoder
+Pb
Free Decoder
Probe? Back to 2008
Sample Dilutions To Point4 Select a
Plates To Point Select b
Auto
This navigational button will take you to where you enter your Test Plate Cq values (see next page):
19.) Once here, enter your values as shown – using the following procedure: Note: first clear both entry fields by activating this button and this button first, and then Copy, Paste Special Values your Test Plate Cqs into their appropriate spots underneath the correct target name: NTC 1 Clear Cqs 2 3 4 5 6
eae 33.20 18.70 21.10 22.30 24.80 26.00 27.50
Z5214 50.00 21.30 23.00 24.80 26.40 27.50 29.00
HKG 50.00 18.50 20.30 20.90 23.40 24.40 25.70
Z5211 38.20 22.70 24.10 25.40 27.80 29.10 30.60
7 8 9 10 11
28.60 30.10 31.30 31.80 32.50
30.90 33.10 34.20 36.30 38.60
27.60 28.70 29.80 31.40 33.00
32.50 33.70 34.90 37.40 36.90
To Home
Eliminated Targets adjust
Clear Cqs
Ctrl Shift C
Add these targets now
To Plate Adjust UMES
ALL DOTS WEIGHED
To Sample Aiming
To The Top
To Point Select b
To Point Select a
SC
(Enter up to 7 more Targets and their Avg. Test Plate Cq values here) Enter "50" if blank
To Printouts
To Greenland
To Quick Access
Ctrl Shift C 20.) Activate the button (this imports your Test Plate data into the point-selection processing work area and automatically throws out “obvious bad points”)… 21.) After the Macro finishes running you then manually select all existing graph data ranges for viewing – graph by graph, and you will see the following images in the first 4 graphs (your 4 targets): ints, then construct curves from the remaining good data in each case
ALL WE
eae
Apparent good Stnd Curve
start useful stnd c
50 40
Efficiency:
30
Correlation:
Ct
110.86% -0.988 b = 15.182 m = -3.0864
20
y = -3.0864x + 15.182 2 R = 0.9753
To Point Select b
10
-8
-7
-6
-5
-4
-3
-2
-1
Ctrl Sh Ctrl+Shift+B t
0 0
[log]
T
Z5214 start useful stnd c
Apparent good Stnd Curve 50 40
84.77% -0.998 b = 15.448 m = -3.7504
Efficiency: Correlation:
Ct
30 20
y = -3.7504x + 15.448 2 R = 0.9957
10 0
-8
-7
-6
-5
-4
[log]
-3
-2
-1
0
All 4
HKG start useful stnd c
Apparent good Stnd Curve 50 40
105.54% -0.998 b = 13.769 m = -3.1960
Efficiency:
30 Ct
Correlation:
20
y = -3.196x + 13.769 2 R = 0.996
To Sample Aiming
10 ALL WEI
0 -8
-7
-6
-5
-4
[log]
-3
-2
-1
0
Z5211 start useful stnd c
Apparent good Stnd Curve 50 40
Efficiency:
30
Correlation:
Ct
100.04% -0.995 b = 17.873 m = -3.3209
20
y = -3.3209x + 17.873 2 R = 0.9901
10 0
-8
-7
-6
-5
-4
[log]
-3
-2
-1
0
See how to select the data ranges, graph by graph on the last page of this document…
22.) Next, you want to eliminate the points that seem to be problematic. You do this by taking note of which point on the graphs it is (of 11 possible points) and then eliminate the corresponding dot in column BJ in this particular file (“TestPlateResultsAnalysis2006.xls”), after which, to incorporate C t rl your deletions (or additions) you will activate the S hif button, and then reselect all existing t B graphical data again (graph by graph) to see the new lines you have obtained. Eventually, repeating this process until you believe what you have, you will arrive at what looks like the good points to accept for each graph. During my point selection process for your Test Plate results, I found the following points to be “acceptable” for each of the 4 targets (so they can fit on this page, I show them side-by-side instead of vertically as they appear in column BJ in the program):
. . . . . . .
. . . . . . . . . .
. . . . . . . . .
. . . . . . . . .
These point selections yielded the following graphs: ints, then construct curves from the remaining good data in each case
ALL WE
eae
Apparent good Stnd Curve
start useful stnd c
50 40
Efficiency:
30
Correlation:
Ct
97.86% -0.995 b = 14.590 m = -3.3743
20
y = -3.3743x + 14.59 R2 = 0.9893
To Point Select b
10
-8
-7
-6
-5
-4
-3
-2
-1
Ctrl Sh Ctrl+Shift+B t
0 0
[log]
T
Z5214 start useful stnd c
Apparent good Stnd Curve 50 40
87.80% -0.998 b = 15.725 m = -3.6536
Efficiency: Correlation:
Ct
30 20
y = -3.6536x + 15.725 R2 = 0.9968
10 0
-8
-7
-6
-5
-4
[log]
-3
-2
-1
0
All 4
HKG start useful stnd c
Apparent good Stnd Curve 50 40
106.61% -0.999 b = 13.953 m = -3.1731
Efficiency:
30 Ct
Correlation:
20
y = -3.1731x + 13.953 R2 = 0.998
To Sample Aiming
10 ALL WEI
0 -8
-7
-6
-5
-4
[log]
-3
-2
-1
0
Z5211 start useful stnd c
Apparent good Stnd Curve 50 40
Efficiency:
30
Correlation:
Ct
92.29% -0.998 b = 17.308 m = -3.5216
20
y = -3.5216x + 17.308 R2 = 0.9956
10 0
-8
-7
-6
-5
-4
[log]
-3
-2
-1
0
after which I:
23.) Found a common good dynamic dilution range within which to work – a region common to all targets. For convenience of visualizing this region, use the available moveable red- or blue-dotted guidelines (which you can click on and pull/grab manually from the sides of the graphs and pull to corral the user-friendly range that you think looks good for all targets). These lines have already been pulled into position as shown in the previous image – and they indicate a dilution range from 1:100 to 1:10,000; which to me, seemed to be a nice range for all four of these targets. Also, other inductive points of concern here – and/or things to think about: are whether or not you 1.) will run out of any particular sample working within this range, and 2.) whether or not the more dilute ends of the curves bend downward (which the first and last targets did) – therefore possibly indicating non-specific product formation(s) (something seen more predominantly with primerdimers in SYBR Green-based qPCR, but can also be seen here with off-target influences to an extent with probe-based qPCR) but we side-step these problem regions by choosing to work within a more concentrated sample dynamic range (between the dotted red lines), and 3.) whether or not you have witnessed any inhibitory behavior of your sample mixture serially tested on your Test Plate. To me it appears as though there is very little to no inhibitory behavior in your sample prep (which is great), so, the next line of concern here is to find out whether or not one or more of your samples is not plentiful (concentrated) enough to work within the chosen range of “workship” here… But, before we check into that, we first tell the program about the “1:100 to 1:10,000” range we want to use and work within…: 24.) First go to the TestPlateResultsAnalysis2006b.xls file by activating the near the top of the TestPlateResultsAnalysis2006.xls file (near cell BZ10). This button can be found elsewhere in the program as well. Once there, which is a nearly-identical twin-sister file to the file we just left:
To P o int Select b
button
25.) Select all graph ranges as you did in the first twin file, and pull the red or blue lines to the desired working range (like before), then: enter “35” into cell BY1 to tell the system there is little to no inhibitory behavior noticed even near your most concentrated Test Plate dilution (1:33.33). “35” is a close enough friendly number is why 35 was chosen. 26.) Enter the desired working range values (“100” and “10000”) as shown here and here and 1 here: adj.upper: 1.070062 Stnd. Curve Ctrl Shift Z adj.lower: 0.205934 eae "in-well" start useful stnd curve at 1: 100 100.0 serial 1: 4.641589 464 2154 Efficiency: 97.86% 118.36% 10000 Correlation: -0.995 (Better E) b = 14.590 4 points adjust dil: 1 To 2008b m = -3.3743 f 1 des.start: 100 ALL 3-pt. Lift 1 des.end: 10000 same as 1st 1 . 4-pt. Get desdilfact: 1 Then 27.) Activate the “ALL same as 1st” button to use this range for all targets… And then activate the Lift button (which will paste these values into the appropriate cells in UMES.xls file – see next page step 30 – but perform steps 28 and 29 first):
28.) Look in or near UMES.xls cell H55 and activate the navigational button:
To Sample A iming
Once there… To Quick Access
29.) Clear cell BB29 and activate the navigation button:
near cell AW25…
30.) In this region: The “Lift” button entered “10000 here (cell H49) and “100” here (Cell I49) Stratagene 1-STEP
if OFF
Stratagene 2-STEP
Hi
Choose Standard Amounts: C 100 uL of each standard
sample #1
Lo
10000 10000
Approx. cost: $815.71
No. of Plates: 10 S4 S3 Mo>
Achieved
(uL) COMPREHESIVE SERIAL DILUTION TABLE (uL) Total made
A B C D E
Stock I
Water
to Next
(FINAL VOL.)
Dilutions 1:
177.2
59.1
118.1
77.2
100 uL
3
154.4
77.2
77.2
54.4
100 uL
6
126.2
54.4
71.8
26.2
100 uL
13.9247664
121.5
26.2
95.4
21.5
100 uL
64.63303976
100.0
21.5
78.5
100 uL
300
Actual Final Dilutions Achieved for Final Plates after used in-well:
1: 1: 1: 1: 1:
100 200 464.15888 2154.434659 10000
Now, to make the value “59.1” better, I entered “65” in cell M2 and activate the “Activate” button (then wait for about a minute) … This gives you a more “beauteous” amount to work with here out of the gate when preparing the standards from your Stock I material … Voila!: Achieved
(uL) COMPREHESIVE SERIAL DILUTION TABLE (uL) Stock I
Water
to Next
(FINAL VOL.)
Dilutions 1:
195.0
65.0
130.0
84.9
110 uL
3
169.9
84.9
84.9
59.8
110 uL
6
138.9
59.8
79.0
28.8
110 uL
13.9247664
133.8
28.8
104.9
23.7
110 uL
64.63303976
110.1
23.7
86.3
110 uL
300
Total made
A B C D E
Actual Final Dilutions Achieved for Final Plates after used in-well:
1: 1: 1: 1: 1:
100 200 464.15888 2154.434659 10000
In this same worksheet in cell region Q53:T59 are found the factors to enter into the machine to inform it of the standards’ relative strengths: Machine factors eae 1 0.5 0.215443471 0.046415889 0.01
Z5214 HKG Z5211 1 1 1 0.5 0.5 0.5 0.215443 0.215443471 0.215443 0.046416 0.046415889 0.046416 0.01 0.01 0.01
36.) Catch the nearest star Sample Dilutio ns
Activate the button:
home and: here Ctrl m
~ C.H. Hidden VERSION rendered 5-10-2010/2-20-2011. Dedicated to my family…
Ctrl Ctrl x ISURF #03407 Shift F
Property of ISU-ISURF
TP Cq
Reset
To Quick Access
To Printouts
Ctrl w LCM Add
MMRT
To LCM Adjust To Top
Simple Mix
Primer File
SC To Sample Aiming
Go To Questionnaire
To ERROR List
Inhib. Thresh
Quanta To MM Decoder
+Pb
Free Decoder
Probe? Back to 2008
Sample Dilutions To Point4 Select a
Plates To Point Select b
Auto
37.) Print out what you see there … these are the dilutions of your samples, that when used in your qPCR reactions, will attain the “red-dot” dilution you have chosen. (These are the normalizing dilutions already sent to you in your final set up pdf)… as shown on the following page here too:
Additional dilutions of the individual (1:5) 35 samples to attain red-dot dilution in qPCR eae To Printouts Aux Tier 1 1:5 DNA Sample Water 1st total 1 15.3 uL 184.7 uL 200 uL Cust Dil. 2 13.5 uL 186.5 uL 200 uL 3 10.4 uL 189.6 uL 200 uL 4 54.1 uL 145.9 uL 200 uL 5 15.6 uL 184.4 uL 200 uL 6 71.4 uL 128.6 uL 200 uL 7 48.1 uL 151.9 uL 200 uL 8 85.4 uL 114.6 uL 200 uL 9 28.5 uL 171.5 uL 200 uL 10 47.0 uL 153.0 uL 200 uL 11 39.9 uL 160.1 uL 200 uL 12 39.5 uL 160.5 uL 200 uL 13 60.3 uL 139.7 uL 200 uL 14 61.1 uL 138.9 uL 200 uL 15 60.1 uL 139.9 uL 200 uL 16 82.1 uL 117.9 uL 200 uL 17 102.3 uL 97.7 uL 200 uL 18 106.3 uL 93.7 uL 200 uL 19 66.5 uL 133.5 uL 200 uL 20 80.9 uL 119.1 uL 200 uL 21 49.3 uL 150.7 uL 200 uL 22 48.2 uL 151.8 uL 200 uL 23 39.5 uL 160.5 uL 200 uL 24 39.8 uL 160.2 uL 200 uL 25 70.7 uL 129.3 uL 200 uL 26 74.6 uL 125.4 uL 200 uL 27 42.0 uL 158.0 uL 200 uL 28 36.2 uL 163.8 uL 200 uL 29 53.5 uL 146.5 uL 200 uL 30 31.7 uL 168.3 uL 200 uL 31 53.3 uL 146.7 uL 200 uL 32 22.2 uL 177.8 uL 200 uL 33 48.9 uL 151.1 uL 200 uL 34 8.7 uL 191.3 uL 200 uL 35 19.8 uL 180.2 uL 200 uL
The “red-dot” dilution in terms of “ng/uL” is always shown in UMES.xls cells E1 and J27 and also appears elsewhere in beguiling fashion inside of the “zPrintouts.xls” file tab “Qspecs” worksheet in cell range C3:I3 and here, there and throughout cell range Y1:AW156 in that worksheet, e.g.: 0.2352 ng/uL DNA achieved in-well for each different qPCR Target: Z5214 HKG Z5211 eae 0.23521 0.23521 0.23521 0.23521
*
Not used Total pg of DNA per eae Z5214 4704.2 4704.2
Absolute Q Not used
eae
Z5214
HKG
Z5211
1120047.6
1120047.6
1120047.6
1120047.6
~0.0042 pg DNA/cell
Est. # of cells' DNA/well:
20.00 uL rxn: HKG Z5211 4704.2 4704.2
and Tier 1
0.23521 ng/uL eae 1: 200.00 (dilution of Tier 1)
Sample "Aiming" Device 7.0 6.0
Tier 1
5.0 4.0
= 0.23521 ng/uL
3.0 2.0 1.0 0.0 -2.5
-2
-1.5
-1
Indiviual Target Abs. Attenua
-0.5
0 in-well
Tier 1 eae Scroll 1 Tier 1 Target
eae
dil factor between samples
surrogate
ng/uL
LOG input
DCt values
Abs. Template
2.3207944
464.15888
0.215443471
-0.666666664
2.214618719
4.6415888
2154.434659
0.046415889
-1.333333327
4.429237439
0.47042 0.23521 0.101349 0.021835
4.641588901
10000
0.01
-2
6.64385619
0.004704 ng/uL
0
100
1
0
0
2
200
0.5
-0.301029996
1
ng/uL ng/uL ng/uL ng/uL
38.) Now we want to attain our Final Plates Mastermix output. So, similar to before (when attaining the Test Plate Mastermix output): First catch the nearest star
Ctrl m
~ C.H. Hidden VERSION rendered 5-10-2010/2-20-2011. Dedicated to my family…
Ctrl Ctrl x ISURF #03407 Shift F
Property of ISU-ISURF
TP Cq
Reset Auto fill 7 like 1st
To M M Deco der
to home position, and, once there, activate the To Quick Access
To Printouts
Ctrl w
MMRT
To LCM Adjust To Top
Simple Mix
Primer File
SC To Sample Aiming
Go To Questionnaire
LCM Add
To ERROR List
Inhib. Thresh
Quanta To MM Decoder
+Pb
Free Decoder
Probe? Back to 2008
Sample Dilutions To Point4 Select a
Plates To Point Select b
ENTER TARGET AND FINAL IN-WELL [nM] PRIMER-PROBE INFO Fix Species Target Name Fwd [nM] Rev [nM] Probe [nM] eae 250 250 bact Z5214 250 250 bact HKG 200 200 bact Z5211 250 250 bact
button
And, once at the destination, which should look like the following: GOOD
FIX
FIX2
20.00 uL prepared/Well 20.00 uL used per Well Depends on Machine/MM used
SYBR?:
Target Master Mix Set-up(s) 480.00 uL 0.00 uL 288.00 uL 768.00 uL
2X Master Mix: 0X RT-Taq Solution: Water: Total MMRT prepared:
1
Total MMRT needed:
6.00 uL
split
Rev primer:
0.00 uL
into
Probe:
6.00 uL
12
MMRT: 192.00 uL 0.00 uL water: 3
0.00 uL Copy to Free MM Decoder
To Free MM Decoder
then add
Attain xtra 2
0.00 uL
Z5214
More
xtra made
Less
X4
Reduce Xtra
X4
More
17.00 uL amounts
Extra/Target
Less
Sample Plate(s)
To Printouts
0.00 uL
RNA added/well:
Main Primer-Probe nM
Desired xtra samples prepared
0
93.5 uL
Master adjust: T 6.00 uL
split
Rev primer:
0.00 uL
into
Probe:
6.00 uL
12
MMRT: 192.00 uL water:
wells prepared
1 Round
3.00 uL 48 48 1 1 1.45833333
0.00 uL
250 nM FWD primer 0 nM REV primer
Well size prepared: 20.00 uL
Fwd primer:
3.00 uL Sample to each
xtra ea.made
Total MM ea. 204.00 uL
768.00 uL
eae Fwd primer:
Attain ALL xtras
0.00 uL
Test Plate Final Plates
17.00 uL amounts then add
NO EXTRA
3.00 uL Sample to each
To NRC
4
Enter up to 44 targets
5/10/2010
1 2 3
eae Z5214 HKG
4 5 6 7 8 9 10 11 12 13 14
Z5211
NRC target
To NRC -375 suggested xtra entry Reduce
Since we are seeking the Final Plate set-ups at this point in this manifesto, we merely activate the Final Plates button here to send PQ off on its fetch mission to get the Final Plate mastermix NO parameters. After activating this button, activate the button here and then … EXTRA
Copy to Free MM Decoder
39.) Activate the button. This copies all things over to the “Free Mastermix decoder” (“qPCRUniversalPQMixDecoder12-1-09.xls”) file (which has universal flexibility to accommodate pretty much any master mix on the planet).
We must again inform this naïve file of the Multiplex approach by: 40.) Altering the Target entry area to tell it, effectively, that we are running only 1 target, (although we know it is 4 targets at the same time). We do this by entering only one target name into the region in cells L2:L5 that now look like this: Enter up to 44 targets
5/10/2010
1 2 3
eae Z5214 HKG
4 5 6 7
Z5211
NRC target
But we now alter it to look like this: Enter up to 44 targets
5/10/2010
1 2 3 4 5 6 7
ALL4
NRC target
41.) Then enter your desired multiplex primer-probe usages inside the “StrengthFactors” tab (worksheet) inside this file (but your values should already be here from doing your Test Plate):
Go ahead and activate this tab to go to this sub-worksheet…
Inside this tab (worksheet) your particular entries should already look like: Multiplexing:
Number of targets?
Desired final Final sought mixture volume Stock nM nM 500 uL 526.31579 uL 10000 250 FWD: 10000 0 REV: 10000 250 Probe:
5 "strength factor" 5.000000000000000000X
62.50 uL 0.00 uL 62.50 uL
Target 1
10000 10000 10000
250 0 250
FWD: REV: Probe:
62.50 uL 0.00 uL 62.50 uL
Target 2
10000 10000 10000
200 0 200
FWD: REV: Probe:
50.00 uL 0.00 uL 50.00 uL
Target 3
10000 10000 10000
250 0 250
FWD: REV: Probe:
62.50 uL 0.00 uL 62.50 uL
Target 4
FWD: 0.00 uL REV: 0.00 uL Probe: 0.00 uL Water: 25 uL Total mix: 500 uL collective nanomolarity
Target 5
10000 10000 10000 No of oligos:
8 1900
Proof of final nM FWD 250
Use
Go Back
250
Probe
250
FWD
250
Probe
200
FWD
200
Probe
250
FWD
250
Probe
Eliminate water
Once you have confirmed the red values are correct, activate the “Use” button. TOP Then activate the button there which should be somewhere in your field of view… This should bring you to the following scene (see the next page):
GOOD
FIX
To SF
Target Master Mix Set-up(s)
20.00 uL used per Well Depends on Machine/MM used
SYBR?:
Total MMRT prepared:
1
oligo mix used
ALL
328.00 uL
ignore
41 uL ea. of 8 oligos split
ignore
0.00 uL
into
ignore
0.00 uL
41
MMRT: 1066.00 uL 0.00 uL 0.00 uL
xtra made
Attain xtra
0.00 uL
Well size prepared:
20.00 uL
RNA added/well:
3.00 uL
-20
Desired xtra samples prepared
41
wells prepared
82
144.5 uL
Master adjust: 2 1/2X
RESET
Fwd primer:
1 Round
1 NO EXTRA 1
1.10365854 1
Rev primer: Probe:
Sample Plate(s)
MMRT:
5X oligo mix used
water:
6.00 uL Sample to each
xtra ea.made
Total MM ea. 1394.00 uL
0.00 uL
2X
34.00 uL amounts then add
Extra/Target
1066.00 uL
Total MMRT needed:
328.00 uL
water:
820.00 uL 0.00 uL 246.00 uL 1066.00 uL
2X Master Mix: 0X RT-Taq Solution: Water:
20.00 uL prepared/Well
Attain ALL xtras
Custom xtra
42.) Calculate extra amounts made (safe extra so you don’t run out of mastermix during setting the reactions up physically in the lab; adjust cells I5 and O13 file and then activate: to Attain xtra attain the desired parameters. Work with these settings awhile until you arrive at satisfactory values for safe extra amounts made. You will also see that you can type the desired extra amounts directly into cell H4 for extra amount left over after filling all tubes with primers-probes/mastermix, and cell O12 for how much extra initial master mix you have before adding the primers-probes to it. These values are exactly attained by again activating the Attain xtra button. And, to round up the primer-probe value to the nearest whole integer (for ergonomic, pipetting-related reasons), you can activate the button to Round attain that. There are other convenient functionalities here to tweak to infinity … but you can easily discover them on your own (the buttons are named somewhat intuitively here and there)…
Important: in cell P36 of this file, you must now account for your 4 additional control-type reactions by adding 4 to the number of samples tested. 35 + 4 = 39; enter 39 in cell P36 as shown below:
Add 4 to 35 = 39
2 39 1 5 x 90 90
Technical replicates: Number of Samples: Number of Targets: Points in Stnd Curve: NTC wells used?: Wells per target: Total wells:
Adjust Target No.
0 To NRC
Don’t forget to do this … So: The final amounts that I arrived at for this is: GOOD
FIX
To SF
Target Master Mix Set-up(s)
20.00 uL used per Well Depends on Machine/MM used
SYBR?:
Total MMRT prepared:
1
oligo mix used
ALL
390.00 uL
ignore
split
ignore
0.00 uL
into
ignore
0.00 uL
45
MMRT: 1267.50 uL 0.00 uL 0.00 uL
xtra made
Attain xtra
127.50 uL
-20
Well size prepared:
20.00 uL
RNA added/well:
3.00 uL
Desired xtra samples prepared
45
wells prepared
90
114.5 uL
Master adjust: 1.083333333 2 1/2X
RESET
Fwd primer: Probe:
Sample Plate(s)
MMRT:
5X oligo mix used
water:
And shown again below (with Final Plate set-up drawing):
Round
1 NO EXTRA
1.08333333 0.99215686 1
Rev primer:
6.00 uL Sample to each
xtra ea.made
Total MM ea. 1657.50 uL
50.30 uL
2X
34.00 uL amounts then add
Extra/Target
1267.50 uL
Total MMRT needed:
48.7 uL ea. of 8 oligos
390.00 uL
water:
1013.69 uL 0.00 uL 304.11 uL 1317.80 uL
2X Master Mix: 0X RT-Taq Solution: Water:
20.00 uL prepared/Well
Attain ALL xtras
Custom xtra
Now, to inspect whether or not there is sample inhibition or whether your have run out any sample at this point, catch a star home, and then activate the “Inhibition Thresh” button here Ctrl m
~ C.H. Hidden VERSION rendered 5-10-2010/2-20-2011. Dedicated to my family…
Ctrl Ctrl x ISURF #03407 Shift F
Property of ISU-ISURF
TP Cq
To Quick Access
To Printouts
Ctrl w
To LCM Adjust
SC To ERROR List
Inhib. Thresh
Quanta
+Pb
Free Decoder
Back to 2008
Sample Dilutions Point4
To MM Decoder
Probe?
To Top
Simple Mix
Primer File
Reset
LCM Add
MMRT
To Sample Aiming
Go To Questionnaire
To Select a
Plates To Point Select b
Auto
Then activate the
Inhibitio n Repo rt
button once you are transported to the destination…
You will now be taken to UMES.xls cell region AF1:AH36 – and you will see that no inhibition is expected from any of your samples as they attain their in-well “red-dot” dilution during final qPCR assessment. This is all based on the behavior of your sample mixture (Stock I) on your Test Plate… and so far, with numerous qPCR projects in different parts of the world – to date, using PQ, this has proven to be a good assumption every time. The only better way to do this is to make a standard curve out of every single sample for every target. Now to inspect how much of each 1:5 sample you have left after completing the whole run – inspect UMES.xls cell range E75:H11 and also, in cell F26 is shown the limiting sample volume among all of your 35 samples. There is an Error messaging system built into the program – the older PQ Manual explains that somewhat. (Below explains steps 21 and 22 further):
First click on a graph Then you will notice that the data that makes up the x and y ranges is boxed in
Click/grab and pull this corner of the blue box downward or upward (whatever the case may warrant) to stretch the boxes over the entire range of data displayed. Right now, only data range BO2:BP4 is displayed in the graph, but we want to see all of the data: BO2:BP8. As shown below…
See below
Now the data range for this graph has been selected correctly. Do this manually for each graph… (I can’t find a way to tell Excel to do this automatically, yet). ~Jack ~
