Bench work
- Miniprep (done with Daniel Catt)
- Follow instructions for miniprep
- measure concentration of DNA
- store @ -20°C
- Forgot to make glycerol stocks
- after spinning cells, but before re-suspending them, streaked a plate with samples of each cell pellet.
- → 37°C ~6h
- Glycerol stock cultures (done by Daniel Catt)
- 3 mL LB + 3μL 100 mg/mL Amp + V(His)+I colony #1 cells from plate in step 1 above
- 3 mL LB + 3μL 100 mg/mL Amp + V(His)+I colony #2 cells from plate in step 1 above
- 3 mL LB + 3μL 100 mg/mL Amp + V(His)+I colony #3 cells from plate in step 1 above
- 3 mL LB + 3μL 100 mg/mL Amp + V+I colony #1 cells from plate in step 1 above
- 3 mL LB + 3μL 100 mg/mL Amp + V+I colony #2 cells from plate in step 1 above
- 3 mL LB + 3μL 100 mg/mL Amp + V+I colony #3 cells from plate in step 1 above
- Sequencing
- send miniprep samples for DNA sequencing: pTXB1His+BSA colony #1 and pTXB1+BSA colony #2
- Use T7 promoter and terminator universal primers
Results
- Miniprep on samples worked without issue:
Results of miniprep: DNA quantitation
| Sample (20x dilute) |
A1 (Abs @ 260nm) |
A2 (Abs @ 280nm) |
Ratio A1/A2 |
[DNA] @20x dilution (μg/mL) |
[DNA] @1x (μg/mL)
|
| pTXB1+BSA Colony 1
|
0.263 |
0.141 |
1.8644 |
13.135 |
262.7
|
| pTXB1+BSA Colony 2
|
0.638 |
0.330 |
1.933 |
31.9 |
638
|
| pTXB1+BSA Colony 3
|
0.541 |
0.278 |
1.946 |
27.05 |
541
|
| pTXB1+His+BSA Colony 1
|
0.350 |
0.183 |
1.913 |
17.5 |
350
|
| pTXB1+His+BSA Colony 2
|
0.310 |
0.156 |
1.987 |
15.5 |
310
|
| pTXB1+His+BSA Colony 3
|
0.397 |
0.202 |
1.965 |
19.85 |
397
|
- Samples sent off for sequencing with T7 and T7 term primers
(7μL of sample + 7μL sterile dH2O):
- pTXB1+BSA Colony 1
- pTXB1+His+BSA Colony 1
- Kathryn Muratore 15:27, 19 July 2011 (EDT): Sequencing reactions did not work. pTXB1+BSA with T7 promoter primer had some readable sequence, but a BLAST shows only a short homology with adenovirus.
|
AU CHEM-570 Lab Prep
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