about assembly heavy chain and light chain of mouse ScFv

[Allison]

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).

Good luck.
Allison


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!
> 
> Bo
> 
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