genepool

Next Generation Sequencing and Bioinformatics
at the University of Edinburgh


Methods used by the GenePool

Sanger Sequencing: Sequencing Protocol.

For BigDye Terminator Cycle Sequencing ready Reaction Kit.

Quarter reactions master mix

                          1 sample      1 plate        2 plates    3 plates
   5X Sequencing Buffer     2  µl       212 µl         424 µl        636 µl
   ddH20                    3.68 µl     390 µl         780 µl       1170 µl
   Primer (10 µM)           0.32  µl     34 µl          68 µl        102 µl
   BigDye                   2  µl       212 µl         424 µl        636 µl

Eighth reactions master mix

                          1 sample        1 plate        2 plates    3 plates
   5X Sequencing Buffer     2  µl         212 µl         424 µl        636 µl
   ddH20                    4.68 µl       496 µl         992 µl       1488 µl
   Primer (10 µM)           0.32  µl       34 µl          68 µl        102 µl
   BigDye                   1  µl         106 µl         212 µl        318 µl

108 µl of master mix in each strip well, transfer 8 µl to each well

DNA template 2 µl in each well

Cycles

   Slow cycle            Fast Cycle
   95°C 30sec            96°C    1min
   50°C 20sec            96°C    10sec
   60°C 4min             50°C    5sec
   60°C 1min 15sec
 
   25 cycles
   hold at 4°C

1 Defrost the BIGDYE, primers and DNA and keep on ice. After defrosting ensure each component is mixed thoroughly by gentle pipetting.
2 Place a clean 0.2 ml PCR tube on ice.
3 Add Sequencing Buffer and ddH20 to the tube.
4 Add 2 µl BIGDYE
5 Add primer.
6 Add 2 µl clean PCR* product. Mix well by gentle pipetting.

* Recommended template amounts

   PCR product:     100-200bp    1-3ng
                    200-500bp    3-10ng
                   500-1000bp    5-20ng
                  1000-2000bp    10-40ng
                      >2000bp    40-100ng
   single-stranded               50-100ng
   double-stranded               200-500ng

7 Place tube onto PCR block.
8 After cycle is complete remove and store at 4°C.


The GenePool has core support from: GenePool admin site
edinburgh
The University of Edinburgh
School of Biological Sciences
The Darwin Trust
of Edinburgh
darwin
mrc
nerc
bbsrc
sulsa

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This is version 2.2 of the GenePool website (July 2009).