8 replies to this topic

[suulei]

I'm doing PCR to verify transgenic plants. First time I set up a PCR 3 samples with both positive and negative controls. It is  pretty good! But the second time when I set up a large scale PCR with 50 samples, I got no PCR products at all ! including the positive control.
I'm sure that the system is right, I didn't miss any component. the mineral oil was added both time and the programs are same except hot lid was used the second time.
I want to know does the hot lid increase the annealing temperature, thus ruin my PCR?

[hula]

I don't think so. hot lids are widly used nowadays. Did you use the same PCR machine?

Hula

[suulei]

I used the same machine and the same program.

[lararid]

suulei, on Jun 17 2003, 04:40 AM, said:

I'm doing PCR to verify transgenic plants. First time I set up a PCR 3 samples with both positive and negative controls. It is  pretty good! But the second time when I set up a large scale PCR with 50 samples, I got no PCR products at all ! including the positive control.
I'm sure that the system is right, I didn't miss any component. the mineral oil was added both time and the programs are same except hot lid was used the second time.
I want to know does the hot lid increase the annealing temperature, thus ruin my PCR?

The only role of hot lid is to prevent evaporation. Pcr machines have an uniformity in block temperature. So something else is wrong. Good luck!

[zingmatter]

There are 2 things to remember:-

(1) PCR stands for Polymerase Crap Reaction (i.e. it didn't work because the moon was in the wrong phase, or you had the wrong coloured socks on etc...)

(2) There's a car bumper sticker that goes - "Molecular biologists do it again and again and again and ... ..."

To summarise, there you probably didn't do anything wrong, you just have to do it again. If it fails again then consider changing you stocks of dNTPs and primers - they're usually the first to go.

Hope this helps

zing

[gio]

I Know that hot lid is to pevent evaporation so use mineral oil is not necessary, i do alla my PCR without mineral oil and hot lid...try it!

[fei]

If you have use hot lid,don't add mineral oil and try again

[jadefalcon]

I got a similar problem some time ago...
Test PCRs with only some samples (3-5) were working fine, the follwoing larger scale PCR, 45 samples, came up with no PCR products at all. Since everything was the same (Machine, program, enzyme, dNTPs, etc.), i was quite frustrated...

So i split the large scale up in two 22 sample "shifts" and - it worked. So while pipetting the 50 samples, the time it took to set up the PCR was responsible for something to go wrong. Since then i do only 25 samples max at the same time and everythings fine....
Though i have no clue at all what it could have been, but as long as it works...

Mike

[tfitzwater]

L. Mezei  used the lambda DNA control in Perkin Elmer’s GeneAmp Reagent Kit to analyze the effect of using the oil overlay (reactions in triplicate). (L. Mezei, 1990 Amplifications 4:11-13.) Yield with oil 2013 ng and CV of 6%.  Yield without oil 405 ng and CV of 72%.

Unless you are using a machine specifically designed to be used without oil, it is risky to leave out the oil.  Evaporation causes the top of the reaction volume to cool as much as 2°C. (L. Haff and J. Atwood 1989 Amplifications 3:15-16.) This can result in amplification artifacts. Samples without oil fail to reach the denaturing temperature.  Due to evaporation, the concentration of components gradually increases.  The magnesium concentration has a dramatic effect on enzyme processivity and the resulting error rate.  

Percent of water evaporated from a 100 µL PCR reaction without oil overlay: cycle 5/12% evaporative loss, cycle 10/13% evaporative loss, cycle 15/28% evaporative loss, and cycle 20/39% evaporative loss.  Mezei also determined that the optimum amount of mineral oil was 50-70 µL.  Oil volumes above or below this range resulted in a drop of at least 1°C in annealing temperature.