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Digestion of PMP and PGL4.30 for ligation
prepared PMP2 , PGL2 and PGL3 for ligation
restriction enzyme digest mixtures were as follows:
PMP2 control for plasmid
- DNA 2µL
- HindIII 1µL
- XbaI 1µL
- N4 buffer 10x 5µL
- BSA 100x 0.5µL
- Water 40.5 µL
PMP2 control for NotI
- DNA 2µL
- NotI 1µL
- N4 buffer 10x 5µL
- BSA 100x 0.5µL
- Water 41.5 µL
PMP2 control for SpeI
- DNA 2µL
- SpeI 1µL
- N4 buffer 10x 5µL
- BSA 100x 0.5µL
- Water 41.5 µL
PMP2 for ligation
- DNA 19µL
- SpeI 1µL
- NotI 1µL
- N2 buffer 10x 5µL
- BSA 100x 0.5µL
- Water 23.5 µL
PGL2 for ligation
- DNA 12µL
- HindIII 1µL
- MfeI 1µL
- N2 buffer 10x 5µL
- BSA 100x 0.5µL
- Water 31 µL
PGL3 for ligation
- DNA 16µL
- HindIII 1µL
- FseI 1µL
- N2 buffer 10x 5µL
- BSA 100x 0.5µL
- Water 26.5 µL
All let 1:30 hours at 37ºC, shaking (600 rpm).
After we ran the digestion products in a gel (1% agarose, 50 µl of digestion product + 10 µl of loading dye).
| ladder | ' | PMP2 cut w/ NotI SpeI | ' | PGL2 cut w/ HindII MfeI | ' | PGL3 cut w/ HindII FseI | ' | control of HindII w/ PGL3 | control of FseI w/ PGL3 |
| ladder | control w/ HindIII and XbaI for PMP2 | Control of NotI w/ PMP2 | Control of SpeI w/ PMP2 | eGFP pcr product | TNFR pcr product | SEAP pcr product | |||
The gel fragments corresponding to the linearized backbones in wells 3, 5 and 7 on the top comb were cut out with a razor blade. The PCR products in wells 8, 10 and 12 on the bottom comb were also cut out.
The gel fragments were run through a Qiagen gel purification kit.
Protocol: None
Forgot to insert protocol.
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