Crude genomic lysate was prepared according the Crude Genomic Lysate for PCR protocol of the dd1-8a, dd5-Q, dd8-63a, dd11-2a,dd12-22a, and the roach trachea isolates. Primers 8F and 1492R (16S rRNA) were used in a PCR reaction using the lysate as DNA template. Amplicons were ran on a 1% gel (seen below). 10 uL of amplicon + 2 uL EZ Vision loading dye. Hi Lo DNA marker was used.
| Lane1 | Lane2 | Lane3 | Lane4 | Lane5 | Lane6 | Lane7 | Lane8 |
| Hi Lo | dd8-63a | Roach | dd5-Q | dd11-2a | dd12-22a | dd1-8a | H2O |
Discussion:
It turns out that the issue I have been having with blank gels can be linked to an issue with the DNA template since there were amplicons on the gel that was ran above. Ampliconsc an be observed in lanes 3 - 6, but not in lanes 1 and 7. The Hi Lo DNA marker isn't very clear, which is odd since I can usually determine fairly easily where the bands are located. It appears that the amplicons appeared around the 1500 bp marker, not unusual for a 16S rRNA gene. I am not certain why no bands appeared in Lanes 1 and 7. Perhaps there was an issue with the crude genomic lysate that was used, maybe there wasn't enough template, since there should have been amplification.
In conclusion, it appears that the issue I was having was with the genomic DNA isolation kits, and that the primers and PCR parameters are functioning appropriately. I will next use a different DNA isolation kit to extract genomic DNA and then run PCR.
//EWW
I think something happened w/ your lane labels. What is in lanes 2-8?
ReplyDeleteAlso, you should probably do a proper gDNA prep for this. Not just a crude lysate.
ReplyDeleteYeah, i haven't fully updated that blog post yet. I have the lane labels, I just didn't put it on their yet. I know i need to do a proper gDNA prep, but we were having some issues with the kits and i just wanted to make sure that the DNA was really the problem and not the primers/PCR. And it turns out it was indeed an issue with the kit
ReplyDelete