DNA sequencing Sample Preparation

What to bring?

The following types of DNA are suitable for sequencing. Please provide the specified amount of DNA:

  • double stranded DNA - 0.7-1.0 µg total
  • single stranded DNA - 0.2-0.4 µg total
  • cosmid, BAC - 2.0-5.0 µg total
  • pcr fragments:

100-200 bp 10 ng

200-500 bp 10-20 ng

500-1000 bp 20-50 ng

1000-2000 bp 50-100 ng

Please notice the following requirement for purity

  • 260/280 = (1.8-2.0)
  • 260/230 > 1
  • A micro spectrophotometer is available at the Unit for testing samples of DNA for sequencing (NanoDrop). Please notice that due to extensive workload the use of this spectrometer is restricted only to DNA samples that are submitted for sequencing .

Volume

The total volume of the reaction should be:

  • 15 microliters when we add the primer.
  • 20 microliters when the sample already includes the primer. (If the DNA is concentrated, please dilute it in water up to the right volume)
  • Please bring the sample in 0.6 ml tube.
  • For more then 40 samples you can prepare them on 96 well pcr microplate (0.1 ml). In each well put 10 microliters (total) contain half amount of the DNA and half amount of the primer.

   The samples should be from A1 - H1, A2 - H2, etc. (the rows are of 8 samples each).

   When you pay for the each plate, please come to the DNA Sequeincing unit (Levine building, room 203), and bring the order number without entering the sequence information.

   At the unit we have a special program for entering the sequence information.

  Please bring your plate between 8:00 - 15:00, when a unit memeber is there.

Primers

Available primers from the DNA Sequencing Facility

T3 5' - GCG CAA TTA ACC CTC ACT AAA G - 3'
T7 5' - TAA TAC GAC TCA CTA TAG GG - 3'
KSi 5' - CCT CGA GGT GGA CGG TAT CG - 3'
SK 5' - CGC TCT AGA ACT AGT GGA TCC - 3'
M13-REV 5' - AAC AGC TAT GAC CAT GAT TA - 3'
M13-20 5' - TGT AAA ACG ACG GCC AGT - 3'
PUC REV 5' - AGC GGA TAA CAA TTT CAC AC - 3'
PET REV 5' - GCT AGT TAT TGC TCA GCG G - 3'
SP6 5' - CCA AGC TAT TTA GGT GAC AC - 3'
SP6 19 (for pcdna3) 5' - GAT TTA GGT GAC ACT ATA G
Poly T T(24)
  • Internal primers should be at least 18 bases with 50% GC (Tm=50 deg)
  • Please provide 10 pmol of primer per reaction.

What not to bring

  • samples with salt (260/230 is low)
  • samples with ratio 260/280 out of range
  • samples with RNA
  • samples with slow growing host vectors -jm101
  • samples with ethanol or phenol still in them

When to bring samples

The DNA Sequencing Facility is open 5 days a week between 08:00 - 16:00  for bringing the samples. People who need help can  call us at #3688.

After working hours you can put the samples in the mail box, outside of the Levine building.