Thursday, June 25, 2009
- Checked plates (ROB and NORA) of SKY4182 (Longtine plasmids pFA6a-GFP-KanMX) left a few more hours at 32C.
=>Few more cells grew, but no noticeable colonies in both plates. Gave to Satoe to put in 37C incubator upstairs for awhile to see if we can get cultures
- Checked 6 3mL culture grown from giant bacteria blob last night at 37C in LB-Amp. All are cloudy. Will prep with the group.
ROB 1,2,3
Nora 1,2,3
- Since only 4 people, will mini-prep ROB 1,2 and NORA 1,2 and check with restriction digest.
- 30μL mini-preps
- Ran on gel with 2μL DNA. Dark band at ~5kb as expected.
- Took concentrations with Qbit fluorometer, calibrated using DNA BR buffers and reagents.
- Incubate standard and DNA samples (diluted 100-fold) for 1 minute before taking measurement)
ROB1: 700μg/mL
ROB2: 132μg/mL
NORA1: 431μg/mL
NORA2: 840μg/mL
- All have good concentration. Restriction digest with BamHI and BglII (should see a ~1kb band along with `4kb fragment)
Restriction digest
- Digest ROB1,2 and NORA1,2 along with -DNA and -Enzyme controls
+DNA
NEB buffer: 1μL
BSA (10X): 1μL
DNA:2μL
Enzyme: .5+.5μL
ddH2O: 5μL
-DNA
NEB buffer: 1μL
BSA (10X): 1μL
DNA:0μL
Enzyme: .5+.5μL
ddH2O: 7μL
-Enzyme
NEB buffer: 1μL
BSA (10X): 1μL
DNA:2μL
Enzyme:0μL
ddH2O:6μL
- 10μL total reactions
- Incubate for 1hour in 37C water bath. Run all 10μL on agarose gel (+2μL 6X dye).
- Digest of NORA 1,2 (left) and ROB 1,2 (right) with 1kB ladder, -DNA control (far right, not visible) and respective -Enzyme controls
- Reading wells left to right:
- 1kb ladder
- NORA/ROB 1
- NORA/ROB 1 -Enz
- NORA/ROB 2
- NORA/ROB 2 -Enz
- -DNA
- Gels look fine but this was technically a test-rune. Cultures should be made with single colonies. If our plates that Satoe put back into the incubator show colonies tomorrow, we'll retry this on Saturday with cultures taken from single colonies
- Hopefully primers will come tomorrow and we can start PCR of Longtine plasmid
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