6 replies to this topic

[Indhumathi]

Hi all,
I am in the first year of my PhD and I am facing a great deal of problem with PCR. Mock PCR with sterile water also gives positive result with exact size of my product (around 120 bp). Is it possible to have such a persistent contamination problem to get same size band always. I cloned single inserts which is around 120 bp. the real problem came up when I tried to clone two inserts which should have given 250 bp band. But it also came around 120 bp even after so many repeats. Then I went for mock PCR with sterile water which also gave the same result. What could be the problem? does hairpin loop in primers cause self priming and lead to amplification in PCR?
Indhu

[metionina]

Just to understand:
1. You want to clone a fragment of 120 pb in a plasmid. When you do your PCR to confirm cloning, you have a band around 120 pb. But, is it your insert? Did you sequence it? Did you try a digestion of your plasmid? What is the problem?
2. Your mock PCR shows a band around 120 pb. Are your solutions contaminated? Try to change all your solutions (primers, Taq, buffer, water...).
3. What kind of gel do you use for the migration of your PCR? 120 pb is a small fragment.
3. What is the problem with 2 inserts?

[Indhumathi]

Hi metionina.thanks for the reply
1.actually, I ve not tried sequencing it. but if we try digesting the plasmid, can we see the difference with such a small fragment?
2.I actually changed our primers and premix, but still encountered the same problem.
3.usually I use 1.5% gel for smaller fragments and when running the mock PCR I used 2% gel.
4.the problem in 2 inserts is that am not sure whether the two inserts are getting ligated or the single insert is getting cloned, since for both am getting the band around 120bp.
I understand that sequencing is the better option but I want to rule out the PCR problem first before going for sequencing.
Indhu

[metionina]

1. you can try to digest your plasmid+insert with an enzyme recognizing a site present only in your insert: only one digestion, only one site in the insert (do you have it?). If your plasmid can linearize you can be sure you have your insert in your plasmid.
2. do a PCR with primers that hybridize on your plasmid just after and before your insert. If you have an amplification you have an insert.
3. use a positive positive control (a PCR of your insert before ligation). Just to be sure that the band you see on the gel is your insert. Use 2% gel and a short time migration. Sometimes primers can be visible on the gel and you see them as a kind of band below 200 pb. Do you use a good marker for small fragments?
4. in order to be sure to have 2 inserts in your plasmid, use primers that hybridize on your plasmid (as point 2 so you can have a 240 pb PCR) or digest your plasmid (2 digestions before and after your insert to linearize the plasmid and show a 240 pb fragment (do not cut in inserts)

[Ameya P]

Hi Indhu,

I faced a similar problem a few months ago. I would suggest running a No template control in the following manner to rule out that the band you see is not because of the primers itself.

• NTC with only forward primer
  
• NTC with only reverse primer
  
• NTC with both forward and reverse primer.

If you see a PCR product around 120 bp in tube 3, you know the problem is with the primers. Here, you would have to change the primer sequence in a way that this does not repeat.

Otherwise, hope that one of Metionina's suggestions give you some information.

Good Luck

[Indhumathi]

hi al,
sorry for the late response. I found out the real problem was with our primers. I have got a new set of primers synthesized and have finished the first set of cloning. thanks for your suggestions.
Indhu

[Trof]

Ameya P, on 17 September 2012 - 12:29 AM, said:

Hi Indhu,

I faced a similar problem a few months ago. I would suggest running a No template control in the following manner to rule out that the band you see is not because of the primers itself.

• NTC with only forward primer
  
  
• NTC with only reverse primer
  
  
• NTC with both forward and reverse primer.

If you see a PCR product around 120 bp in tube 3, you know the problem is with the primers. Here, you would have to change the primer sequence in a way that this does not repeat.

About this setting I wonder. What is the purpose of testing the NTC with one primer only? Unless the single primer is contaminated with the other primer there would be no amplification even if the primer is contaminated by template DNA.
Or does this test assume DNA contamination (by a PCR product possibly) is combined with contamination by the other of the primer pair as well?
Or am I missing something? (if we are not of course talking of a single-primer dimers, but that wouldn't be called false positive, because it's different product)