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From 2008.igem.org

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Revision as of 12:43, 14 July 2008

1)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

2)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)

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