Re: Elution retarded band - (Mar/23/2001 )

T/A vector works fine for this purpose. You can even make T/A vector yourself.

Hans

Hi Hans and Long,

Thanks to both of you for your helpful suggestions. I will try out TA cloning. Actually I want to sequence my RT-PCR product. But right now I am trying direct sequencing method for the PCR product. Hope this works!

Abhijit.

Hi friends,

I have been facing some problem with cloning of my 500 bp RT-PCR product. I used a Stratagen kit for this purpose. A month back I had used this same kit to clone a 200bp insert successfully, but not this time. The vector is Amp Script SK (+).. that is a modified pbluescript vector with a Srf I site inserted in the MCS. I have followed the protocol supplied with the kit but to no avail. I would like some suggestions or tips to troubleshooting from those who have faced this kind of problem and are familiar with these techniques. Lots of good wishes to all of you.

Abhijit.

I don't know what specific purpose of your pcr cloning, sequencing or expression? I agree with Hans, TA cloning is fine. I usually use TA cloning kit from Invitrogene and never failed. If you want to express your pcr products you can also use TA cloning. Many companies also provide expression TA cloning kit.

Good luck

Long

How do you make T/A vector?

You can find several protocols under Molecular Biology>DNA>TA cloning.

Longthe webmaaster

Hi,Have you double-checked to make sure that your new RT-PCR product doesn't have Srf1 site anywhere? If it does have it, you can just design primers with unique restriction sites and cut them before ligation. Best wishes.

PC

Hi,Have you double-checked to make sure that your new RT-PCR product doesn't have Srf1 site anywhere? If it does have it, you can just design primers with unique restriction sites and cut them before ligation. Best wishes.

PC