about assembly heavy chain and light chain of mouse ScFv
allison at nospam.com
Wed Jul 6 09:00:23 EST 2005
When I was have trouble with the assembly reaction I talked with tech
support at Amersham. The person I spoke with suggested using less linker
primer in the first step of the assembly reaction. I finally got a
linked band when I used 50ng each of H and L chain with 2uL of the
linker primer mix (instead of 4uL). I kept the volume of RS primer the
same in the second step of the reaction (4uL).
bo yang wrote:
> Hello, everyone,
> I am now doing mouse ScFv antibody by using Amersham Mouse ScFv Module
> Phage Antibody System. However, I can not link the heavy chain and light
> chain. I tried optimizing the overlapping PCR conditions such as
> changing template amount, denaturing temperature, decreasing RS primer.
> However, I still did not get ScFv fragment. I am wondering what the most
> important problems in this step are?
> Your help will be greatly appreciated!
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