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Team:ESBS-Strasbourg/Restriction digestion
From 2008.igem.org
(→b) Protocol) |
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-qsp H2O 100µL <br> | -qsp H2O 100µL <br> | ||
->digestion (see table) | ->digestion (see table) | ||
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'''double digestion protocol used for plasmid verification 17/07/08 (KK):''' | '''double digestion protocol used for plasmid verification 17/07/08 (KK):''' | ||
*10µL of preped plasmid (around 1µg) | *10µL of preped plasmid (around 1µg) | ||
Revision as of 13:27, 18 July 2008
a) Remarks
| Name | Digestion time | Reaction temperature | Buffer to use (100%) | Heat inactivation | Unit number |
| EcoR1 | 2h | 37°C | NEBuffer 1/2/3/4 | 65°C, 20 min | 0,13 |
| Spe1 | 2h | 37°C | NEBuffer2+100µg/mL of BSA | 65°C, 20 min | 0,5 |
| Xba1 | 2h | 37°C | NEBuffer2+100µg/mL of BSA | 65°C, 20 min | 0,13 |
| Not1 | 2h | 37°C | NEBuffer3+100µg/mL of BSA NEBuffer2 (only 50%) | 65°C, 20 min | 0,25 |
| Pst1 | 2h | 37°C | NEBuffer3+100µg/mL NEBuffer 1/2 (only 75%) | 80°C, 20 min | 0,5 |
| Dpn1 | 1h | 37°C | NEBuffer 1/2/4 | 80°C, 20 min | 0,13 |
NB motto: the less unit number an enzyme has, the stronger activity she own
b) Protocol
Two different strategies could be considerate:
-In the case of a step-by-step digestion process, always begin to digest with the enzyme the less efficient
-In a double digestion case, check of course the buffer to use, and the activity of the two enzymes must be very similar. Maria don’t support this strategy so much, however we can try it and check if we have good result (it could be usefull for the Spe1 and Xba1 digestion of course)
Furthermore, Maria use to do digestion during 2h, then she adds once again 1µL of enzymes for another digestion time of 2 hours. She says its very efficient, but I find it quite long (four hours…) Discussion to elaborate the best strategy has to be hold
standard method:
-30µg DNA
-20 enzyme unit (5u/µL), of each enzymes
-10µL tampon RE 10X
-10µL BSA 10X
-qsp H2O 100µL
->digestion (see table)
double digestion protocol used for plasmid verification 17/07/08 (KK):
- 10µL of preped plasmid (around 1µg)
- 2µL Tango buffer (10x)
- 0.2 µL BcuI (SpeI) - two times the quantity which is necessary
- 0.2 µL XbaI - two times the quantity which is necessary
- 7.6 µL H20
This protocol has to be up scaled, if you want to go on working if the digested fragement
