Hey everyone,
I used degenerate primers to create PCR products. They showed a single strong band on the gel.
First I tried to send them in with the degenerate primers, but -as expected- it was not possible to sequence them this way.
Then I tried doing blunt-end cloning using the pJET cloning kit by Thermofisher and transformed the plasmids into E. coli DH10beta. It worked really good for the positive control in the Kit but it did not work with my PCR products. All of the plasmids that I prepared from the colonies had been empty according to the size of the control PCR (~200 bp). I already tried to incubate them longer in the ligation step (30 min) and I use a higher ratio (double the recommended amount).
Has anyone any tip for this Kit (I'm the first person in my working group to use it) or a different way to sequence my PCR product?
Hi Verena,
Just wanted to let you know that if your PCR products were amplified using Taq DNA polymerase, then you need to purify your PCR amplicons followed by making them blunt before cloning into a blunt end vector. Else you can directly clone purified amplicons in a T/A or U/A cloning vector. After cloning and plasmid DNA miniprep, you can amplify and sequence the insert using vector-specific primers.
best of luck,
SD
I did sequence HCV NS5 sequences using my primers which have degenerate bases and it worked well.
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