Sequencing PCR products
imnr at connectnet.com
Thu Jul 6 15:34:48 EST 1995
In article <3tc599$cs1 at server.st.usm.edu> jstemple at whale.st.usm.edu (Jeffrey Shane Temple) writes:
>From: jstemple at whale.st.usm.edu (Jeffrey Shane Temple)
>Subject: Sequencing PCR products
>Date: 4 Jul 1995 19:37:13 GMT
>I am planning to use a relatively new product from Amersham to try and
>sequence PCR products. The name of the product is Sequenase PCR product
>sequencing kit. Before I buy the kit, I would like to have some feedback
>from others that may have used this kit before. Is it as easy as the
>catalog describes it and most importantly, is it worth $300? If you
>haven't used this kit before and have another suggestion, feel free to
>voice it. All comments will be greatly appreciated.
>Univ of Southern MS
>Dept of Chem/Biochem
I haven't used that particular kit, but I have a modified Sequenase protocol
that has worked well in our lab for directly sequencing PCR products. Use the
Sequenase Version 1.0 kit. The crucial part is the concentration of the primer:
Put 1ug PCR product in 8ul water, add 2ul 2N NaOH, incubate at room temp. for
Add 7ul primer (0.5 O.D.260/ml or 15ng/ml)
Add 3ul 3M NaOAc and 75ul cold ethanol
Precipitate at -70C for 20 min., spin 20 min.
Rinse pellet with 70% Ethanol, air dry (you may not see the pellet, don't
Resuspend in 8ul water(from here on it's pretty much your standard Sequenase
protocol) Add 2ul 10X Sequenase buffer
2ul 1:5 Extension mix
1ul 0.1M DTT
1ul Alpha S35 dATP
2ul 1:8 Sequenase
Incubate at room temp. 5 min.
Aliquot 2.5ul each G,A,T,C into appropriate tubes(prewarm to 37C)
Add 3.5ul seq rxn to each tube, 37C 10 min.
4ul stop buffer, boil and load
Hope this helps and saves you some money.
Immune Response Corp.
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