Tag Archives: 1.5t

PCR – Dungan isolates from 20090402 with Euk primers

Did PCR with new Euk primers designed by Steven. Should be one step higher taxonomically. PCR set up is here. Aneal temp 50C.

Lane 1 – Hyperladder

Lane 2 – 19t

Lane 4 – 13t

Lane 6 – 17t

Lane 7 – 100bp ladder

Lane 8 – 1.5t

Lane 10 – 1.2t

Lane 11 – Hyperladder

Lane 12 – 11t

Lane 14 – H5

Lane 15 – 100bp ladder

Lane 16 – 12t

Lane 18 – H2O

Lane 19 – H2O

Laen 20 – Hyperladder

Results: The new EukA/B primers worked wonderfully. The brightest band in each lane was excised and purified using Millipore DA spin columns. These will be stored and sequenced at a later date.

PCR – New Dungan isolates

Repeat of PCR from 20090403, but using AmpliTaq and 50C annealing temp. PCR set up is here.

Lane 1 – Hyperladder

Lane 2 – 19t

Lane 3 – 100bp ladder

Lane 4 – 13t

Lane 6 – 17t

Lane 8 – 1.5t

Lane 9 – Hyperladder

Lane 10 – 1.2t

Lane 12 – 11t

Lane 13 – 100bp ladder

Lane 14 – H5

Lane 16 – 12t

Lane 17 – 100bp Ladder

Lane 18 – H2O

Lane 19 – H2O

Lane 20 – Hyperladder

Results: Nothing amplified! Possibly due to age of polymerase (?); over a year old. Will wait to repeat for new primers to arrive (EukA/B).

PCR – New Dungan isolates

Ran PCR with GoTaq on the new Dungan isolate gDNA from yesterday. PCR set up is here. 53C annealing temp.

Lane 1 – Hyperladder

Lane 2 – 17t

Lane 4 – H5

Lane 6 – 12t

Lane 7 – Hyperladder

Lane 8 – 1.5t

Lane 10 – 1.2t

Lane 12 – 11t

lane 13 – Hyperladder

Lane 14 – 13t

Lane 16 – 19t

Lane 18 – H2O

Lane 19 – Hyperladder

Lane 20 – H2O

Results: All the bands present are a bit larger than 400bp. However, the bottom band in the H5 sample is larger than any band present in any other samples. Additionally, the largest band in the H5 sample is between 800-1000bp. Bands were cut out from H5 (two bands: Top, Bottom), 1.5t, 1.2t, 13t. These will be purified and sequenced.

It’s also interesting to note that the bands present in this gel are found in the same samples as the last time this analysis was done. See Kevin’s Notebook, 20090212.