Tag Archives: MIE-14y

PCR – Two new Dungan isolates from earlier today

Set up PCRs on:

MIE-14y

VNTc-1.2-C1/G10

Used Euk A/B and LABY A/Y primers. Anneal temp 50C. PCR set up is here.

NOTE: Due to the extremely low concentrations of gDNA from these two samples, I used a large amount of gDNA in the rxns. Check the PCR set up link for actual numbers.

Lane 1 – Hyperladder (5uL)

Lane 2 – VNTc-1.2-C1/G10 (Euk primers)

Lane 3 – MIE-14y (Euk primers)

Lane 4 – H2O (Euk primers)

Lane 5 – H2O (Euk primers)

Lane 6 – VNTc-1.2-C1/G10 (Laby primers)

Lane 7 – MIE-14y (Laby primers)

Lane 8 – H2O (Laby primers)

Lane 9 – H2O (Laby primers)

lane 10 – 100bp Ladder

Results: Nada. Probably because of low [gDNA], but could also be due to PCR inhibitors in the gDNA. Will retry using Amplitaq and less gDNA.

gDNA Isolation – Two new Dungan isolates

Isolated gDNA from the following two samples:

MIE-14y

VNTc-1.2-C1/G10

Samples were spun @ 16,000g @ RT for 2mins. No visible pellets in either sample. EtOH was removed. “Pellets” were washed in 1x PBS (pH=7.6) two times and then the Qiagen DNEasy Kit protocol was followed. Samples were incubated @ 55C with Proteinase K for ~2hrs.

Results: Both samples show really, really low quantities of gDNA.