about assembly heavy chain and light chain of mouse ScFv

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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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:

[desertrose]

Dear colleagues
I am having a similar problem,I am working in a project for construction of synthetic scfv,the Ab was tailored by the PI who is abroad now.
I have the Vh and Vl now but I can't do the assembly with linker,
Please could anyone help or tell me how the design is made or suggest some readings or softwares
thanks

[desertrose]

I need help in assembly,i can't do it at all although i have both vl &vh

[desertrose]

Any suggestions, protocols or readings