September 13, 2018

From OpenWetWare

September 13, 2018

FGDS Telomere Length Assay Troubleshooting Pt. 2

Context: I have completed 5 successful plates for the FGDS project. I anticipate I will need to run 18-19 plates total. I started to notice aberrations beginning with the housekeeping half of plate 6. During this plate there was an odd spike in fluorescence which quickly declined again. The samples then amplified normally around cycle 25. Overall that plate was excellent. However, the telomere half for plate 6 was a complete failure. One standard failed to amplify at all and all remaining samples were marked by a strange spike in fluorescence between cycles 15 and 20, after which they seemed to amplify normally around cycle 21-25. Concerned the problem was with the UNG (I had just switched to a new batch), I quickly reset things to rerun the telomere half. This one failed even worse, with many samples failing to amplify at all. Then I left for the ISPNE conference.

Upon my return I conversed with Sue and decided to test my telomere primers. All of my samples had been stored in the 4C fridge, and Sue thought my primers may have been contaminated and begun to degrade. To test this I created a new primer 10uM dilution using the factory stock, which was also stored at 4C. I ran this plate on Tuesday September 11, 2018. Nothing amplified.

Based on this I have two hypotheses for the problem:

  • my sybrgreen stock has gone bad
  • my primers have been degrading for the duration of the time they were stored at 4C, this includes the factory stock as well as the 10uM dilution.

To test this I am running a half plate today using 50 total wells in two groups of 25. The first 24 will be the TL standards along with 3 control samples using telomere primer dilutions I borrowed from Sue. If these do not amplify the problem is my Sybergreen. If these do amplify the problem is my primers. The next 24 will be the 36B4 standards along with 3 control samples using my current dilutions of the 36B4 primers and a new batch of Sybergreen. I’m concerned these might have similarly degraded. If this set does not amplify my problem has spread to all primers. If this does amplify AND the TL does not my problem is the Sybergreen. If this does not amplify and the TL does, my problem has spread to the 36B4 primers.

If nothing amplifies I’m basically screwed.