Protocol for DNA sequencing

Protocol for DNA sequencing Sample preparation

PCR product A.

B.

Amplify the gene section that you are sequencing using the appropriate primers Run PCR product on a gel and record the gel

Plasmid A. B.

C. D.

E.

DNA cleanup after PCR

A. • • • • B.

DNA concentration

Isolate Plasmid from bacteria ( see DNA extraction notes) Use your standard plasmid extraction method. Make sure that the DNA is very clean. If you are using a column cleanup kit please elute in water. Run plasmid in gel to determine isolation success and approximate concentration Plasmid can be sequenced directly

Clean up PCR product to remove primers, NTP’s Taq and primer di-mers (See DNA cleanup methods) Columns such as sold by Qiagen/Roche and other companies can be used. Make sure that you elute in water and NOT in elution buffer You can use EtOH precipitation ( See Sequencing precipitation - this has to be modified to adjust to the volume of your PCR reaction) You can use PEG precipitation – remember you will lose anything that is smaller that 200bp in size. Sephadex columns Run cleaned PCR on gel. Determine if the primers have been removed and verify that you did not loose your DNA during the cleanup process You can also determine the DNA purity and concentration using a spectrophotometer or an Nanodrop

DNA concentration should be : 60 -100 ng per 1000bp for both PCR product and plasmid.

• You need to remember however that you may have to optimize as every persons DNA is different and every gene is different. • If you are sequencing a plasmid you have to calculate the total plasmid size that you are sequencing and NOT only the size of your insert when you are determining how much DNA you need for the cycle sequencing reaction

For a standard quarter reaction DNA x ul 60-100ng Big Dye 2 ul NOTE: Primer x ul 3.2 pmol DNA concentration and Sequencing buffer (5X) 1 ul primer concentration may Water x ul need optimization Total 10ul Precipitation sequencing Preparation Protocol Prepare product 3 M sodium acetate anhydrous 3ul NaOAc (3M. pH 4.6) (NaOAc) pH 4.6 62.5 ul Ethanol (99.9%) Cycle sequencing reaction

according to Standard protocol from Applied Biosystems

NOTES:

•

•

Make up a minimum of 100 ml NaOAc in order to buffer the salt correctly to pH 4.6 Only buffer with Acetic acid

(High grade quality) 14.5ul Water

(You can make a master mix if you have multiple samples – make it fresh every time) Make Sequencing samples up to

•

Aliquot you NaOAc and freeze it away ad -20C • Keep one Eppenorf of NaOAc in the fridge. • Before using the NaOAc Let it equilibrate to room temperature and vortex for at least 30 seconds. Do a visual inspection to see if there are any salt crystals before use. Check the lid inside and outside • Discard any old NaOAc

70% Ethanol •

•

Always prepare fresh 70% Ethanol for your precipitation wash If you use cold Ethanol you will precipitate more of your smaller DNA fragments

NOTES:

Other Sequencing cleanup methods

• When removing supernatant after precipitation add 70% Ethanol immediately. This will prevent salt and free nucleotides form Sticking to the tube when the Ethanol evaporates • Remove as much supernatant as possible without disturbing your pellet. As this contains salt and free nucleotides. If to much remains these will dry and stick to the tube causing problems on the sequencer • Two 70% ethanol washes ensure that you do not have salt remaining in the tube. Use 250ul 70% Ethanol. We resuspend samples from the top of the tube and if salt sticks to the top section of the tube we will catch re-suspend it also and it will be leaded on the sequencer. • Dry on a heat block @ 80°C for 3 minutes. Check if all the Ethanol has evaporated and that you cannot smell Ethanol in the tube. Do not over dry. This makes re-suspending your samples difficult

• • •

20ul with water Mix Ethanol/NaOAc solution and add to Sequencing samples 1. Spin in centrifuge for 30 minutes 2. Carefully remove supernatant and immediately add 250 ul freshly prepared 70% Ethanol 3. Centrifuge for 3-5 minutes 4. Remove supernatant and repeat 70% Ethanol wash. 5. Remove supernatant completely 6. Dry samples to remove Ethanol. Do not over-dry. If you cannot smell Ethanol anymore it should be OK OR Make up a master mix of: 60 ul Ice cold EtOH 11 ul SABEX water 2 ul 3M NaOAc pH4.6 Add to Cycle sequencing product and continue as in step 1 to 6 above.

Sephadex coxlumns Cleanup columns available commercially PEG precipitation

NOTES: •

Please bring your samples in 250ul or 500ul tubes to the DNA sequencing lab-

•

Samples must be in the lab before 9h00 on the day of the run

•

If you have booked and do not bring the samples in time or cancel less than 24 hours before the time you will be charged for the spaces that you have booked unless someone can take up that space

•

If you would like a long run for fragments longer than 600bp, please indicate this on the samples sheet when you are booking.

•

If you have problems with your DNA sequencing please ask for help before it becomes a serious problem.