Tag Archives: Dungan isolates

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.

gDNA Isolation – New Dungan isolates

gDNA was isolated from the following using the Qiagen DNEasy Kit:

xCvC-19t (3/26/2009)

xCvE-13t (3/16/2009)

xCvC-17t (3/18/2009)

UNTc-1.5t (3/18/2009)

VATm-1.2t (3/16/2009)

xCvC-11t (3/18/2009)

BC05Ca18c/H5 (3/27/2009)

xCvC-12t (3/16/2009)

500uL was of each sample was transferred to a 1.5mL snap cap tube. The samples were pelleted by spinning 5 mins @ 16,000g and washed 2x w/ 1x PBS pH=7.6. Samples were then processed according to Qiagen protocol. Initial digestion with Proteinase K was 2hrs.

PCR – Dungan Isolates

Samples (in Chelex) were vortexed and heated @ 95C for 30mins with periodic vortexing. Tubes were spun max speed @ 4C for 2 mins to pellet Chelex. Set up PCR using Immomix master mix. Anealing temp. = 56C. PCR set up here.

Lane 1 – 100bp ladder

Lane 2 – xCvC-11t

Lane 3 – xCvC-12t

Lane 4 – xCvC-17t

Lane 5 – VNTc-12-C1/G10

Lane 6 – BC05Ca-18t/H5

Lane 7 – VATm-1.2t

Lane 8 – VNTc-1.5t

Lane 9 – Neg. Control

Results: PCR seems to have worked for some of the samples. The bottom-most band in lanes 4, 5, 6, 9, & 10 were cut out and stored in “Sam’s Misc. -20C Box”. Date is 1/8/2009, since this PCR ran O/N.

PCR – Dungan Isolates

All samples , except xCVC-11t, are already in Chelex. For xCvC-11t, pipetted a shunk of cells/tissue from source tube. Volume of liquid (EtOH) was ~75uL. Added this to screw cap tube containing 300uL of 10% Chelex solution. Vortexed and incubated @ 95C for 30mins. Vortexed and incubated other samples at 95C for 5mins. Set up PCR with AmpliTaq. Anneal temp. = 53C. PCR set up here.

Lane 1 – 100bp ladder

Lane 2 – xCvC-11t

Lane 3 – xCvC-12t

Lane 4 – xCvC-17t

Lane 5 – VNTc-12-C1/G10

Lane 6 – BC05Ca-18t/H5

Lane 7 – VATm-1.2t

Lane 8 – VNTc-1.5t

Lane 9 – Neg. Control

Results: Ladder is degraded and there are no bands in any lane. Will repeat and try to duplicate Steven’s results from 20080916.