Protocols


Reaction Dilution when you are performing your own cycle sequencing reactions

 

 Reg. 1/4 

 1/8 rxn 

 1/16 rxn 

Total Volume

5

10

10

BigDye

2

1

0.5

5X buffer

0

1.5

1.75

DNA

2

2

2

Primer

1

1

1

H2O

0

4.5

4.75

Notes:

If DNA or primer is not concentrated enough, add more DNA and less water.

If you are submitting samples for a cycle sequencing reaction, we will need the following amounts of DNA and primer (a nanodrop measurement may not even get you in the ballpark. Running DNA on an agarose gel and comparing your sample to the DNA standard you run is more accurate):

Primer 3.2 pmoles
plasmid DNA 300 ng
DNA from PCR 3 ng for products under 200 bp
10 ng for products under 500 bp
20 ng for products under 1,000 bp
40 ng for products under 2,000 bp

DNA sequencing works well for PCR amplified products and is usually more effective than sequencing from a plasmid. The PCR product needs to be purified to remove primers and dNTPs. Some of the different methods for purification include; PrepEase, Exo/SAP, Qiagen, and agarose gel purification. Once clean, one effective way to determine DNA concentration of your purified template is to run it on an agarose gel and compare your band intensity with that of a DNA molecular weight standard with known concentrations.

A submission example: if you are submitting a 500bp PCR product for cycle sequencing, please submit at least 4 microliters containing 3.3 ng/μl cleaned PCR product, and in this same sample aliquot, 1.1 pmole/μl primer. We will then take 3 μl of this mix to add to our cycle sequencing mix.

Processing samples on the Illumina HiSeq 2500 DNA Sequencer

If you are planning on submitting samples for Next generation sequencing on the Illumina HiSeq 2500, please contact Edward Wilcox (phone 422-3647; email DNASC@byu.edu). Library construction manuals can be found at www.illumina.com