Bench work
- Glycerol stocks
- 750μL pTXB1His/DH10B QC colony #2 culture from yesterday + 250μL 60% sterile glycerol → tube #70
- 750μL pTXB1His/DH10B QC colony #3 culture from yesterday + 250μL 60% sterile glycerol → tube #71
- 750μL pTXB1/DH10B colony #1 culture from yesterday + 250μL 60% sterile glycerol → tube #72
- → -80°C
- Miniprep O/N cultures from yesterday
- Quantify [minipreps]
- 95μL water + 5μL DNA from miniprep
- NheI digest
- performing sequential double digest, so use optimal buffer for SapI instead of NEB buffer 2
- modify NheI digest protocol as follows
| Tube |
sterile H2O |
10X NEB4 |
100X BSA |
DNA |
NheI (10 kU/mL)
|
| pTXB1His#1
|
34.5 μL |
5 μL |
0.5 μL |
5 μL of 102 μg/mL |
5μL
|
| pTXB1
|
34.5 μL |
5 μL |
0.5 μL |
5 μL of 82 μg/mL |
5μL
|
| BSA PCR
|
24.5 μL |
5 μL |
0.5 μL |
15 μL directly from PCR reaction |
5μL
|
- → 2h @ 37°C
- → 20' @ 65°C (it took awhile to get the block to the right temp, so they were on ice for a bit, then at ~70°C before the block reached 65°C)
- Note: I should have done a PCR cleanup on the BSA PCR sample! I will repeat this. *Kathryn Muratore 10:20, 20 June 2011 (EDT):
- In retrospect, I'm not sure that I even used the correct tube here *Kathryn Muratore 16:26, 20 June 2011 (EDT):
- I did use the right tube, as seen on the gels later that week *Kathryn Muratore 10:33, 28 June 2011 (EDT):
- → store @ -20°C
Results
{{#widget:Google Spreadsheet
|
key=0Aph9WCMwmkfMdF9PNG53aGllblpSLW1zdmpqcVR0TlE
}}
|
AU CHEM-570 Lab Prep
