Hello,

I've had some trouble with my gels, and are pretty much clueless on what to try next. I've troubleshooted this for the last month to no avail and would like to get some input from the community.

Background:

I am having trouble getting through 200 samples of X. I have successfully gotten through 1/2 of the samples with ideal amplification and no smearing, however recently all my gels started to smear.

The only change I made since then was making new primer stocks from 100mM to 5uM. (which is the working primer concentration which has worked successfully for the last few months).

See the images.

Troubleshooting:

1) Using new Taq Mix, (Still Smearing)

2) Using new RNAse Free H20 (Still Smearing)

3) Using old samples as controls which worked perfectly before, (Still Smearing)

4) New Primer 5uM stocks. (Still Smearing).

5) Ordered 100mM primer's from IDT and made 5um working stocks(Still Smearing)

6) Different PCR machine

7) Ran other control PCR's with other primers, worked perfectly.

The second Image, which has the smearing has the following Lanes.

(After Ladder to the right)

1) Sample 18 (NO PRIMERS)

2) No Template (With PRIMERS)

3)Sample 18 (w/ PRIMERS)