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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).
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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 |
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Protocol: None
Forgot to insert protocol.
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