Leang, Ching 1-1
Preparation of Sequencing Reactions for UMass Core Facility
What the sequencing facility needs from you:
1. In a labeled 1.5 ml Eppendorf tube, combine enough template and primer for 2
reactions.
2. The tube should contain 12 ul with enough template and primer for two
sequencing reactions.
a. Each reaction will have a total of 10 ul made up of:
i. 4 ul Terminator Reaction Mix
ii. 6 ul Template DNA and Primer
3. Template DNA:
a. Template DNA solution must be free of exogenous nucleotides and
oligonucleotides.
i. Use the Promega Wizard kits, and the Qiagen kits for cleanup
which work will in conjunction with fluorescent sequencing.
ii. Resuspend in dH2O and not TE or any solution containing EDTA.
b. For a 10 ul reaction provide the following:
i. Single stranded DNA (e.g. M13)
50-100ng
ii. Double stranded DNA (e.g. dsDNA plasmids)
200-500ng
iii. PCR product DNA (purified)
30-90ng
iv. The amount of template DNA used in the reaction will affect the
success and quality of the sequence.
v. Please make sure that your DNA concentrations fall in the middle
of the values gives.
1. i.e. For double stranded DNA such as that in a plasmid, try
to get as close to 350ng as possible.
vi. Keep in mind that the total volume of template and primer will be
6 ul.
c. For unusually long/short templates or any questions regarding template
preparation call (413) 577-1306.
4. Primer DNA:
a. The primers should be at least 18 bases long to ensure good hybridization
and prevent random priming.
b. Add 1 ul of a 3pM/ul soln. of the desired primer to the mix.
i. Have the primer concentration as close to 3pM/ul as possible.
5. NOTE:
a. The template and primer DNA that are used can not contain any EDTA.
b. Complete the sequencing facility form sheet and return it with your
samples.
c. A print out of the sequencing results can be provided, as well as an
electronic copy size so be sure to provide a floppy or zip disk with enough
space for your results.
d. The chromatographs can be accessed with Prizm Edit View (1.1MB)
available from the sequencing facility.