USTC/Notebook/Restriction Digests

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Latest revision as of 16:55, 27 October 2008

Restriction Digests

Materials

DNA; the thing you want to cut. Usually plasmid or PCR product. Measure concentration in Nanodrop beforehand.
Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers).
Appropriate enzymes.
ddH₂O
BSA (100x from NEB)

Procedure

The following volumes apply to a 20µl analytical digest; for larger, preparative digests, simply scale up (eg. for a 30µl digest, use 3µl of 10x buffer, etc)

add the following components to PCR tube:

2µl BSA (diluted to 10X beforehand)
2µl 10x Buffer
appropriate amount of DNA
0.5µl of each enzyme
appropriate amount of ddH₂O to PCR tube. (20µlTotalVolume-(BSA + Buffer + DNA + Enzyme)µl

eg. 20µl-(5µlDNA+2µlBuffer+2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=10µl ddH₂O)

MIX the reaction by stiring
Incubate the reaction at 37*C for 2-4hrs to ensure complete digestion.
Store digest at -20*C or run immediately on gel.

@@ Line 15: / Line 15: @@
 add the following components to PCR tube:
-*2µl BSA (diluted to 10X beforehand)
+*'''2µl''' BSA (diluted to 10X beforehand)
-*2µl 10x Buffer
+*'''2µl''' 10x Buffer
-*appropriate amount of DNA
+*'''appropriate''' amount of DNA
-*0.5µl of each enzyme
+*'''0.5µl''' of each enzyme
-*appropriate amount of ddH<sub>2</sub>O to PCR tube. (20µlTotalVolume-(BSA + Buffer + DNA + Enzyme)µl
+*appropriate amount of '''ddH<sub>2</sub>O''' to PCR tube. (20µlTotalVolume-(BSA + Buffer + DNA + Enzyme)µl
 eg. 20µl-(5µlDNA+2µlBuffer+2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=10µl ddH<sub>2</sub>O)
-*mix the reaction by stiring
+*'''MIX''' the reaction by stiring
-*Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
+*Incubate the reaction at 37*C for 2-4hrs to ensure complete digestion.
 *Store digest at -20*C or run immediately on gel.