Sequencing PCR products

Immune Response 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.

>Jeffrey Temple
>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 
5 min.
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 
panic)
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.
Katy Lundeen
Immune Response Corp.



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