Tag Archives: LABY

qPCR – Lexie’s QPX Temp & Tissue Experiment (see Lexies Notebook 4/26/2011)

Ran qPCR with Lexie’s cDNA samples from this experiment with the following primer sets in order to better evaluate her biological reps:

QPX_SPB_F/R (SR ID: 387, 388)

LABY_A/Y (SR ID: 116, 121)

LABY was run as a potential normalizing gene. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). Samples were run in duplicate and were labeled according to what was written on the tops of Lexie’s cDNA tubes.

Results:

qPCR Data File (BioRad CFX96)

qPCR Report (PDF)

LABY primers worked, but the melt curves don’t look that good. I’ll let Lexie worry about the rest of the analysis.

PCR – “Unknown” Dungans/Lyons

This is a repeat of yesterday’s set up with LABY primers, but with an annealing temp of 53C in hopes of improving the number of amplicons generated from additional samples. See yesterday’s PCR run for info on samples.

Results: Samples were loaded 1-29 and three negative controls from left to right, top to bottom.

The lower annealing temperature clearly resulted in more products. The ~500bp band was cut from each lane and stored @ -20C. All bands will be purified using Millipore spin columns and then sent for sequencing.

PCR – Dungan isolate (MIE-14v) gDNA from 20090708

PCR of MIE-14v just to make sure that we can’t get a product from this sample, despite NanoDrop readings suggesting that there’s no DNA. Used both LABY and Euk primer sets. PCR set up is here. Anneal temp 50C.

Lane 1 – 100bp Ladder

Lane 2 – Euk

Lane 3 – Euk H2O

Lane 4 – Euk H2O

Lane 5 – Euk H2O

Lane 6 – LABY

Lane 7 – LABY H2O

Lane 8 – LABY H2O

Lane 9 – LABY H2O

Lane 10 – 100bp Ladder

Results: Nothing, as expected. Need to devise a new method of isolating gDNA from these “problem” isolates.

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.