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.
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.
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.
