INSTITUTO NACIONAL DE CANCEROLOGÍA-SSA UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO INSTITUTO DE INVESTIGACIONES BIOMÉDICAS - UNAM UNIDAD DE INVESTIGACIÓN BÁSICA EN CÁNCER LABORATORIO DE VIRUS Y CÁNCER
Short Protocol for cytosine methylation detection with the bisulfite method using the EZ DNA Methylation-GoldTM Kit NOTE: It’s a short method of 3 hrs in termociclator; during the preparation of the reagents don’t expose them for much time to the light and prepare it before use as indicate the instructive. Zymo Research recommends using from 500 to 2000 ng of DNA per treatment. Preparation of CT Conversion Reagent: Add 900 μL of water, 300 μL of M-Dilution Buffer and 50 μL of M-Dissolving Buffer to one tube CT Conversion Reagent. Mix for 10 minutes in vortex.
1. Add 130 μL of the prepared CT Conversion Reagent to 20 μL of DNA sample and adjust the total volume to 150 μL with sterile H2O. Mix the sample by flicking or pipetting up and down. For example: SAMPLE
[ng/μL]
DNA (ng)
VOLUME DNA (μL)
CT-CONVERSION R. (μL)
H2O (μL)
TOTAL (μL)
SiHa
592.0
1300.0
2.2
130.0
17.8
150.0
2. Perform the following temperature steps: 98° C for 10 minutes, 64° C for 2.5 hrs, then hold at 4° C. 3. Add 600 μL of M-Binding Buffer to the Zymo-SpinTM IC Columm. 4. Add the sample. Close the cap and mix by inverting several times. 5. Centrifuge at 11,000 rpm for 30 seconds. Discard the flow-through. 6. Add 100 μL of M-Wash Buffer to the column. Spin at 12,000 rpm for 30 seconds. Discard the flow-through. 7. Add 200 μL of M-Desulphonation Buffer to the column and wait for 20 minutes. After the incubation spin at 12,000 rpm for 30 seconds. 8. Add 200 μL of M-Wash Buffer to the column. Spin at 12,000 rpm for 30 seconds. Discard the flow-through. Add another 200 μL of M-Wash Buffer and spin at 12,000 rpm for 30 seconds. Discard the flow-through and spin at 12,000 rpm for other 30 seconds for complete removal of wash buffer residues. 9. Add 20 μL of M-Elution Buffer directly to the column matrix. Place into a 1.5 mL tube. Spin briefly to elute the DNA. The DNA is now ready for analysis. Using 5 μL of recovered DNA for each PCR reaction.
“HPV DNA Methylation in the cervical carcinogenesis”
By M. en C. Pedro Rosendo Chalma 1
INSTITUTO NACIONAL DE CANCEROLOGÍA-SSA UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO INSTITUTO DE INVESTIGACIONES BIOMÉDICAS - UNAM UNIDAD DE INVESTIGACIÓN BÁSICA EN CÁNCER LABORATORIO DE VIRUS Y CÁNCER
PCR amplification of Bisulfite modified DNA using Taq Gold polymerase PCR conditions by Mina Kalantari: Oligonucleotides designing by Mina Kalantari for LCR amplification. NAME PRIMER SEQUENCING 16msp3F Segment 5’ AAGTAGGATTGAAGGTTAAATTAAAATTTA 16msp3R Segment 5’ AACAAACAATACAAATCAAAAAAACAAAAA 16msp4F Enhancer TATGTTTTTTGGTATAAAATGTGTTTTT 16msp7R Enhancer TAAATTAATTAAAACAAACCAAAAATATAT 16msp5F Segment 3’ TAAGGTTTAAATTTTTAAGGTTAATTAAAT 16msp8R Segment 3’ ATCCTAAAACATTACAATTCTCTTTTAATA
MIX OF REAGENTS: FINAL CONCENTRATION REAGENTS STOCK H2O ------Buffer Taq Gold 10 X 1X dNTPs 1.25 mM 0.2 mM MgCl2 25 mM 1 mM Oligonucleotide 16mspF 10 pMol 1 pMol/μL Oligonucleotide 16mspR 10 pMol 1 pMol/μL Taq Gold Polymerase 5 U/μL 1U TOTAL MIX ------Add 5.0 μL of bisulfite modified DNA. The total reaction is 25 μL.
1X (μL) 5.8 2.5 4.0 2.5 2.5 2.5 0.2 20.0 μL
PROGRAM: STEP 1 2 3 4 5 6 7
TEMPERATURE (°C) 94 94 53 68 Go to 2 rep. 44 68 Hold 4° C
TIME 9:00 0:30 0:30 0:45 7:00
Run 5-10 μL of PCR product on a 2 % agarose gel to verify positives. Cloning of PCR product using TOPO TA kit from Invitrogen. For details see the kit manual.
“HPV DNA Methylation in the cervical carcinogenesis”
By M. en C. Pedro Rosendo Chalma 2
INSTITUTO NACIONAL DE CANCEROLOGÍA-SSA UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO INSTITUTO DE INVESTIGACIONES BIOMÉDICAS - UNAM UNIDAD DE INVESTIGACIÓN BÁSICA EN CÁNCER LABORATORIO DE VIRUS Y CÁNCER
TA Cloning Protocol for PCR products using TOPO TA kit from Invitrogen Time required, 2 hours (protocol written by John Huh, modified by Mina Kalantari), for details see the kit manual. 1) Prepare fresh PCR products. If products aren’t fresh, add Taq polymerase, Mg+, dNTPs mix into each reaction and run at 95° C for 5 minutes and then 72° C for 10 minutes extension to ensure A overhangs are added. 2) In the following order, add to 0.5 tube: Sterile water…………………….. 2.0 μL Salt ………………………………….. 1.0 μL TOPO Vector ……………………. 1.0 μL Fresh PCR product …………… 2.0 μL TOTAL 6.0 μL NOTE: The TOPO Vector must always stay in the -20° C freezer and the TOP10 cells in -80° C freezer.
3) Incubate at room temperature for at least 20 minutes or longer. Aliquot 25 μL or TOP10 cells in a pre-chilled 1.5 mL tubes. NOTE: Be gentile when pipetting TOP10 cells, these cells are very fragile and temperature sensitive. DO NOT refreeze TOP10 cells, once thawed the vial should be completely used.
4) Transfer 4 μL - 6 μL of the complete reaction into each 1.5 mL tube containing TOP10 cells. DO NOT pipette up and down the cells, mix by gentle swirling motion. 5) Keep reaction on ice for 30 minutes. 6) Heat shock: Transfer tubes in a 42° C water bath for 40 seconds. Place tubes back on ice for about 2 minutes. 7) Add 100 μL of room temperature SOC media or LB media in each tube. 8) Place tubes in the 37° C shaking incubator, ~275 rpm, for about 60 minutes. 9) Optional for pCR4 vector, required for PCR2.1 or PCII; add 25 μL of X-gal on each LB + antibiotic plate (Blue/White screening). 10) Place LB + antibiotic (Kan 50 or Amp 100) plates in 37° C incubator or at room temperature. 11) Plate about 50 mL on LB + antibiotic plates in dark 37° C incubator overnight. Observe sterile technique, do not cross contaminate.
“HPV DNA Methylation in the cervical carcinogenesis”
By M. en C. Pedro Rosendo Chalma 3
INSTITUTO NACIONAL DE CANCEROLOGÍA-SSA UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO INSTITUTO DE INVESTIGACIONES BIOMÉDICAS - UNAM UNIDAD DE INVESTIGACIÓN BÁSICA EN CÁNCER LABORATORIO DE VIRUS Y CÁNCER
Gene specific colony PCR and colony streaking 1) Remove all plates from dark 37° C incubator and check for colonies. 2) Prepare colony PCR master mix: Use Taq (any Taq -Promega-) with M13 or specific primers. REAGENTS H2O Go Taq Flexi Buffer dNTPs MgCl2 Oligonucleotide M13F Oligonucleotide M13R Go Taq DNA Polymerase TOTAL MIX
STOCK ---5X 1.25 mM 25 mM 10 pMol 10 pMol 5 U/μL ----
FINAL CONCENTRATION ---1X 0.2 mM 1 mM 1 pMol/μL 1 pMol/μL 1U ----
1X (μL) 11.75 5.0 4.0 2.0 1.0 1.0 0.25 25.0 μL
3) Number 10 colonies. Take a sterile pipette tip (without filters), pick one colony, and ensure that the 2 sides of the tip touches the same colony at least once. 4) Emerge entire tip in the PCR reaction mix. 5) Swirl pipette tip in PCR reaction mix, remove, discard, and cap PCR tubes. 6) Run PCR reaction program: STEP 1 2 3 4 5 6 7
TEMPERATURE (°C) 94 94 60 72 Go to 2 rep. 30 72 Hold 4° C
TIME 7:00 0:30 0:30 1:00 7:00
7) Run 5 μL of PCR product on a 2 % agarose gel to verify positives. 8) Place colony plates in dark 37° C incubator overnight. 9) Save all plates: wrap with parafilm and store in 4° C refrigerator.
“HPV DNA Methylation in the cervical carcinogenesis”
By M. en C. Pedro Rosendo Chalma 4
INSTITUTO NACIONAL DE CANCEROLOGÍA-SSA UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO INSTITUTO DE INVESTIGACIONES BIOMÉDICAS - UNAM UNIDAD DE INVESTIGACIÓN BÁSICA EN CÁNCER LABORATORIO DE VIRUS Y CÁNCER
Purifying of PCR products using SAP and ExoI of USB 1) PCR clean-up: in a 0.2 mL tube, place: SAP Buffer 10X ………………………….……… 1.0 μL SAP …………………………………………….…….. 1.0 μL ExoI…..………………………………………………. 0.5 μL PCR-DNA (80-200 ng) ……………………….. 9.5 μL RUN: 60 minutes to 37° C, 30 minutes to 72° C and hold 4° C Fundament: Exonuclease I and Shrimp Alkaline Phosphatase, together in a specially formulated buffer, to remove unwanted dNTPs and primers from PCR products. Exonuclease I removes residual single-stranded primers and any extraneous single-stranded DNA produced in the PCR. Shrimp Alkaline Phosphatase removes the remaining dNTPs from the PCR mixture.
-------------------------------------------------------------------------------------------------------------------------If you don't have SAP and ExoI of USB you can use of Fermentas Company (Thermo Scientific), is similar and effective. Technical standardized by Martha Martínez and Pedro Rosendo
Shrimp Alkaline Phosphatase ..……….... 2.0 μL Exonuclease I….…………………………………. 0.5 μL PCR-DNA …………………….…………………….. 5.0 μL RUN: 15 minutes to 37° C, 15 minutes to 80° C and hold 4° C Dates of the reagents of Fermentas: Exonuclease I [Cat. No. EN0582 Supplied with: 10X Reaction Buffer, 20,000 u (20 u/µl)], Shrimp Alkaline Phosphatase (SAP) [Cat. No. EF0511 Supplied with: 10X Reaction Buffer, 500 u (1 u/µl)]
Note: If definitively you don't have ExoI and SAP, you can purify for columns of ZYMO, Invitrogen or QUIAGEN. The kits were tested by Pedro Rosendo and gave good results.
ZR DNA Sequencing Clean-up KitTM of Zymo Research Company. Catalog Nos. D4050 & D4051. The column may be regenerated up to 10X. Date Provider in Mexico: Biosistemas Avanzados S.A de C.V. Contact with Alejandra Campos Cortés. Tel/Fax 56 85 07 15 y 56 85 07 03,
[email protected]
PureLink™ PCR Purification Kits provide rapid and efficient removal of short primers, dNTPs, enzymes, short-failed PCR products, and salts from PCR products. SKU # K3100-02.
QIAquick PCR Purification Kit. For purification of 250 PCR reactions: 250 QIAquick Spin Columns, Buffers, Collection Tubes (2 ml). Cat. No./ID 28106.
“HPV DNA Methylation in the cervical carcinogenesis”
By M. en C. Pedro Rosendo Chalma 5
INSTITUTO NACIONAL DE CANCEROLOGÍA-SSA UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO INSTITUTO DE INVESTIGACIONES BIOMÉDICAS - UNAM UNIDAD DE INVESTIGACIÓN BÁSICA EN CÁNCER LABORATORIO DE VIRUS Y CÁNCER
DNA sequencing reaction 2) Sequencing condition by Mina Kalantari for Exo-SAP DNA In a 0.2 mL tube, place: H2O sterile………………………………………………….……….. 2.0 μL 5X Sequencing Buffer………………………………………….. 2.0 μL BigDye Terminator v3.1 Cycler …………………………... 1.5 μL Primer M13 5.0 pMol………………………………………….. 2.0 μL Exo-SAP DNA………………………………………………………. 2.5 μL
If you purify the PCR products with columns, the sequencing reaction is: (Adapted by Pedro Rosendo)
H2O sterile…………………………………………….…………….. 0.5 μL 5X Sequencing Buffer………………………………………….. 2.0 μL BigDye Terminator v3.1 Cycler …………………………... 1.5 μL Primer M13 5.0 pMol………………………………………….. 2.0 μL Columns DNA……………………………………..………………. 4.0 μL
Run sequence: STEP 1 2 3 4 5 6
TEMPERATURE (°C) 94 96 50 60 Go to 2 rep. 24 Hold 4° C
TIME 3:00 0:30 0:15 4:00
“HPV DNA Methylation in the cervical carcinogenesis”
By M. en C. Pedro Rosendo Chalma 6
INSTITUTO NACIONAL DE CANCEROLOGÍA-SSA UNIVERSIDAD NACIONAL AUTÓNOMA DE MÉXICO INSTITUTO DE INVESTIGACIONES BIOMÉDICAS - UNAM UNIDAD DE INVESTIGACIÓN BÁSICA EN CÁNCER LABORATORIO DE VIRUS Y CÁNCER
3) Ethanol precipitate. (You can carry out anyone of the two techniques, both give good result)
Technical 1: DNA precipitation in Freeze process (Written by Kalantari and adapted by Martha Martínez and Pedro Rosendo)
CH3COONa 3.0 M pH 4.6 ……………………………………………….. 3.0 μL Non-denatured 95% Ethanol …………………………………………. 32.5 μL H2O Sterile ………………………………………………………………….... 14.5 μL (Hints… make a large mix and aliquot when needed)
Vortex and precipitated at -20° C Overnight. Spin 25 minutes, max speed at 4° C. Aspirate or pour off supernatant. Wash with 250 μL 70% ethanol, mix and spin 10 minutes at 4°C to max speed. Aspirate or pour off supernatant and dry in Vacufuge (30° C for 20 minutes). Resuspend in 20 μL of Formamida Hi-Di Denature 2 minutes in termoclycler at 96° C, and pour the plaque in ice. Send to sequence. Analyzing of sequence results for methylation sites using the software’s Chromas 3.1 and Lasergene.
Technical 2: DNA precipitation at room temperature using Ethanol for Molecular Biology CH3COONa 3.0 M pH 4.6 …………………………….………………………….. 3.0 μL 95% Ethanol for molecular biology …………………………………………. 62.5 μL H2O Sterile ………………………………………..………………………………….... 14.5 μL (Hints… make a large mix and aliquot when needed) Vortex and precipitated at room temperature for 30 minutes Spin 20 minutes, max speed. Aspirate or pour off supernatant. Wash with 250 μL 70% ethanol for molecular biology freeze, mix and spin 10 minutes at max speed. Aspirate or pour off supernatant and dry in Vacufuge (30° C for 20 minutes). Resuspend in 20 μL of Formamida Hi-Di Denature 2 minutes in termoclycler at 96° C, and pour the plaque in ice. Send to sequence.
“HPV DNA Methylation in the cervical carcinogenesis”
By M. en C. Pedro Rosendo Chalma 7
