W 2/22/12
Goal for this lab: Purify the amplified DNA from first lab, digest it with restriction enzymes, and assess the results with gel electrophoresis.
Zymo Research DNA Clean & Concentrator-5 Cat # T1225 K
$69 for kit of 50
Reagents Per Pair:
- 125 µL Zymo-SpinTM DNA Binding Buffer
- 500 µL Zymo-SpinTM Wash Buffer (GET MORE FOR 2015)
- 1 mL Sterile ddH2O
- 1 Zymo-Spin™ Column
- 2 Capless collection tubes
10 µL 20 mM Spermidine [Should this be 3 tubes of 1 uL??]
1x TAE for 14 gels @ 300 mL/gel = 4200 mL (make at least 5 L)
- 1 2% gel per pair plus 2
- 10x NEB restriction buffer. Students need at most 10µL of any 1 buffer. Check supplies
- Fermentas MassRuler, Low Range
Restriction Enzymes for 2012
Grp | enz 1 | enz 2 | NEB buffer |
---|---|---|---|
ASP | Xho1 | Hind3 | 2 |
BLW | EcoRV | Cla1 | 3, 4, (2) |
CHC | Bgl2 | Cla1 | 3, 4 |
CNT | pvu1 | EcoRV | 3 |
EFO | Hpa1 | Mlu1 | 4, 3 |
KSY | Spe1 | Sph1 | 2 |
LSO | Sal1 | EcoRV | 3 |
NOD | EcoRV | Sac1 | 1, 2, 3, 4 |
RFR | Cla1 | Hind3 | 4,2, |
RMT | Bgl2 | Pvu2 | 3 |
Bgl2 Cla1 EcoRV Hind3 Hpa1 Mlu1 pvu1 Pvu2 Sac1 Sal1 Spe1 Sph1 Xho1
Equipment
- Incubator at 37C, with microtube racks inside
- Water bath at 65C for gels
- copies of semi-log paper (1 per student) (see attached document)
- find powerpoint of semi-log paper and put it on the course website
- Printer-friendly version
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