4 replies to this topic

[Biocat]

In order to control the ligation process, do you think it is efficient to use ligation screening (verify ligation mixture with PCR using vector-specific primers or a combination of vector-specific and insert-specific primers).

Thanks,

Biocat

[fishdoc]

Biocat, on Jun 1 2010, 05:30 AM, said:

In order to control the ligation process, do you think it is efficient to use ligation screening (verify ligation mixture with PCR using vector-specific primers or a combination of vector-specific and insert-specific primers).

Thanks,

Biocat

I only PCR a ligation as a troubleshooting step after a previous ligation doesn't work.

[Biocat]

fishdoc, on Jun 1 2010, 01:50 PM, said:

Biocat, on Jun 1 2010, 05:30 AM, said:

In order to control the ligation process, do you think it is efficient to use ligation screening (verify ligation mixture with PCR using vector-specific primers or a combination of vector-specific and insert-specific primers).

Thanks,

Biocat

I only PCR a ligation as a troubleshooting step after a previous ligation doesn't work.

So PCR a ligation is sensitive enough for confirmation of right ligation.
Should dilute the ligation mixture for PCR...?

[fishdoc]

Biocat, on Jun 1 2010, 07:42 AM, said:

fishdoc, on Jun 1 2010, 01:50 PM, said:

Biocat, on Jun 1 2010, 05:30 AM, said:

In order to control the ligation process, do you think it is efficient to use ligation screening (verify ligation mixture with PCR using vector-specific primers or a combination of vector-specific and insert-specific primers).

Thanks,

Biocat

I only PCR a ligation as a troubleshooting step after a previous ligation doesn't work.

So PCR a ligation is sensitive enough for confirmation of right ligation.
Should dilute the ligation mixture for PCR...?

I've done it many times using between 1 and 5 ul of a ligation reaction as template in a 50 ul PCR. I would imagine it would work diluted, too. I don't know how much you can dilute it, though.

It's usually not a very clean product, but it works.

[geen]

Biocat, on Jun 1 2010, 03:30 AM, said:

In order to control the ligation process, do you think it is efficient to use ligation screening (verify ligation mixture with PCR using vector-specific primers or a combination of vector-specific and insert-specific primers).

Thanks,

Biocat

thnx. can you guide me how to ligate a promoter in any promoterless vector?? i.e., what are the key points which we have to keep in mind andhow can we find out the poromoter boundary i.e., what is the transcriptional start site in any sequence even translational start site AUG is reported. how can we find out transcriptional start site??