Conversation until now:
1) Doing LEAP on affinity purified L1's from PA-1.
"We know: When we do LEAP on affinity purified L1's the activity is much higher than on the sucrose cushion approach - We have done this in the past combining the preps with ~25-50% glycerol and snap freeze them. The activity is maintained - although I don’t recommend long term storage (we saw it does decrease in time). I think this might be a nice addition to your study."
John - "I suspect this may allow you to clone out even more L1 sequences from these RNPs."
2) Including the L1 RNPs affinity purified from a couple different patient tumors to the comparison.
To do eLEAP side by side with endogenous cell lines - this could really bring the impact of the paper up even further.
Jose suggested doing the eLEAP method in NY (but that wasn't possible because we even couldn't fully detect the RNP in the IPs during this time) and sending the amplified products to Spain to clone and secuence.
Jose also suggested:
- Using RNPs from PA-1, from any tumor you may want to test, and even an engineered L1.
- Test using the eLEAP conditions RNPs purified from a wild type engineered and an RT mutant L1 (another control to the study)
John suggested controls like:
- Catalytically dead, mouse IgG, and matched normal tissues
Hua started trying the eLEAP (01/14/19 - Endogenous L1 Element Amplification (eLEAP) assay on PA-1 and MT302) on PA-1 and MT302. It worked, but the MT302 mIgG control was being ampliffied as well. Thus, Jose suggested adding the +heat inactivation negative control.
She tried to do the heat inactivation to get a negative control for the eLEAP (01/22/19 - Endogenous L1 Element Amplification (eLEAP) assay on PA-1 and MT302 with heat inactivation). The inactivation worked, but there was also a small smear around the bands and the MT302 mIgG control was still being amplified, while there was almost no amplification for the PA-1 IPs.
After this they wondered if the problem was the initial amount of powder that was being used (as PA-1 has 10 times less ORF1 than MT302), so she tested different amounts (01/29/19 - eLEAP assay on PA-1 (50 and 100mg scale) with heat inactivation). Here the smear increased and the reaction worked but there were other weird bands as well.
Finally she tried again (01/31/19 - Endogenous L1 Element Amplification (eLEAP) assay on MT302 and 162T / N) However, PCRs were starting to look weirder as she continued. The IgG controls had amplification, also the + heat inactivation samples, and the smear was still there.
- Mixing the parts that seemed to be working (the protocol, amount of beads, mg of powder), I started trying to replicate the 1st and 2nd of her results while doing LEAP in parallel (to check if the RT reaction was working). We also introduced the TurboDNAse digestion, trying to get rid off the contamination:
Both the eLEAP and LEAP seemed to be working, however I also got the smear, so we wondered if the leftovers of extraction buffer from the 1st wash could be interfering with the TurboDNAse reaction. Thus, I tested different kind of buffers to do the wash step previous to the digestion.
The washes using different buffers before TurboDNAse digestion don't seem to be important for improving the reaction - as there is almost no difference between the different samples. The contamination is still there after the digestion.
Regarding the contamination, nothing was amplified on the C- from the 1st PCR, but there is something contaminating the C- from the second PCR. The contamination seems to be something that is amplified when the eLEAP-2 primer is present, in the second PCR cycle. It also contaminated the water from the second PCR.
The contamination probably comes from the sample, as the C- (1st + 2nd), which is the C- (water) used for the first PCR, being used as a C- in the 2nd PCR, wasn't amplified, so the reagents, dNTPs, buffer, enzyme, water and primers seem to be clean. Accidentally, the C- from the 2nd PCR was contaminated during the preparation of the same.