4 replies to this topic

[Lazinase]

Hi everyone,

I'm trying to amplify some patient sample for a specific location. Before doing that, I tested the condition on the lab strain, NL4-3, which gave me a sharp band on the gel. However, when I used the same setting on patient sample, despite of the desired band, there were also some non-specific binding bands...so except increase Tm, decrease primer conc., what else I should do?

Any suggestions is welcome and appreciated.

Edited by Lazinase, 06 May 2009 - 07:33 PM.

[Clare]

Lazinase, on May 7 2009, 04:33 AM, said:

Hi everyone,

I'm trying to amplify some patient sample for a specific location. Before doing that, I tested the condition on the lab strain, NL4-3, which gave me a sharp band on the gel. However, when I used the same setting on patient sample, despite of the desired band, there were also some non-specific binding bands...so except increase Tm, decrease primer conc., what else I should do?

Any suggestions is welcome and appreciated.

Is the quality/quantity of your DNA ok?
Are you sure this 'specific location' is expressed in your patient sample? (if you are doing RT-PCR)
If the PCR conditions work for your NL4-3 PCR I would not go messing around with the PCR conditions.
Clare

[Lazinase]

Hi Clare,

Thanks fo your suggestions. Actually there are four sets of primers I am using on the same patient DNA sample, while three of them give me a sharp band except the last one. So i would think my template DNA is ok.

[leelee]

Is it possible that there are mutations in the patient strain in the primer binding sites??
I did a google for NL4-3, is it a HIV strain- and if so, mutations are quite common no??

[Lazinase]

leelee, on May 7 2009, 08:45 PM, said:

Is it possible that there are mutations in the patient strain in the primer binding sites??
I did a google for NL4-3, is it a HIV strain- and if so, mutations are quite common no??

Well...after getting the sequences back from the other primers, the region that I'm working on seems highly conserved (<5 mutations when I compare to the HIV database)

I think the thing is  the NL4-3 I used as positive control only contains the HIV DNA while the patient sample also contain genomic DNA, which will highly increase the chance of non-specific binding...