9 replies to this topic

[Pratibha Sharma]

Hi

I am in big trouble as my pcr sometimes work but sometimes dont
My reaction is 40 cycle got it from some other lab sometimes i get the pcr product of 1.5 kb but  when I put a reaction again with the same sample it does not give any band only a smear is there . i dont know why i get oit sometimes and sometimes dont .
i can give additional information if needed . this is a long pcr lasts for 5 hours .
pratibha
Ph.d student
AIIMS
New delhi
INDIA

[ocean]

hello
i'm getting the same thing as well...we are testing out our taq just to make sure it's not a doggy batch.  i think a 5 hour reaction is too long, don't u think?

[SVTX]

Hey Pratibha,

Few questions,
1)What kind of PCR machines do you use at AIIMS?  Are they with heated lids?
2) I agree with ocean, 5 hrs is a long time for a 1.5 Kb fragment, unless you are dealing with some forensic sample or the starting material is in low amount.
3) Since you can get the product sometimes, I believe either your region is too sensitive to temperature fluctuations or there is improper mixing of the reaction mix (or some other reason!)
4) What are your conditions for PCR and what is the kind of Taq that you are using?

SV

[Pratibha Sharma]

I agree with you  but this  reaction has exten step of 20 sec at each extension cycle and this is what i am doing from a published paper .its an RT-PCR reaction (rNA - cDNA-PCR)
I got it in the first reaction but now sometimes i get it sometimes its just a smear in the low moleculer weight region .
we are using MJ reasearch dyad machine here and all other PCR reactions are coming ok
i think sample concentrations is a problem

can you suggest what should be the initial RNA concentration for such a reactiuon to work because i always get smears in the low mol weight region

[Pratibha Sharma]

I agree with you  but this  reaction has exten step of 20 sec at each extension cycle and this is what i am doing from a published paper .its an RT-PCR reaction (rNA - cDNA-PCR)
I got it in the first reaction but now sometimes i get it sometimes its just a smear in the low moleculer weight region .
we are using MJ reasearch dyad machine here and all other PCR reactions are coming ok
i think sample concentrations is a problem

can you suggest what should be the initial RNA concentration for such a reactiuon to work because i always get smears in the low mol weight region.
pratibha Sharma
AIIMS

[Charon]

Well if you are making a RT-PCR, what reverse transkriptase are you using?
Depending on speed you might not need extensions. In addition, are you sure that the RNA is ok? If the samples are somehow sheared or partly digested by RNAses you can get smears, too.

[macunha]

Hi Pratibha,

I was reading a news paper when I saw your name. Is it you??? The link is that one: http://www.lexpress.....asp?ida=430951
(click on "Reportage photo" : one the last pictures)

[Ali]

Dear Prathiba

We should take care with contamination in RT-PCR. Are you sure the band that you got first is your target?. Also, you should make control (No RT control=without sample, and mRNA control) to make sure no contaminantion in your RT-PCR. We always do RT-PCR in special place (that can be treat with UV). You can try to use some kits for RT-PCR (for example the SuperScript First-Strand Synthesis System for RT-PCR).

[LabGirl]

I was having the same problem, and I added oil to my samples and the problem disappeared. Our thermocyler has a heated lid, so I didn't think I needed oil, but apparently I was wrong. Might want to try that along with the other good advice....good luck.

[george@CASE]

Change your water. Don't just get the another vial of water from the same batch. Get it fresh from source.