Difference between revisions of "Diagnostic RP Digestion for Checking Insert Size"

Latest revision as of 16:19, 21 June 2010

This procedure is for checking the insert size of a BioBrick part. It uses digestion with EcoRI + PstI.

Label a 1.5 ml tube for each of the digestions to be set up
Transfer about 300 ng into each tube and make up the difference with pure dH2O to 6 ul. Or just use 6 ul of a miniprep
If you have fewer than 4 digestions, add the following to each tube:

  9 ul dH2O
  2 ul 2 NEB4 10X buffer
  2 ul 10X BSA
  0.5 ul HF EcoRI
  0.5 ul HF PstI

If you have 4 or more digestions, prepare a master mix as follows, where N is the number of digestions, then add 14 ul of it to each tube:

  9 x (N+1) ul dH2O
  2 x (N+1) ul 2 NEB4 10X buffer
  2 x (N+1) ul 10X BSA
  0.5 x (N+1) ul HF EcoRI
  0.5 x (N+1) ul HF PstI

@@ Line 1: / Line 1: @@
-'''Diagnostic RP Digestion for Checking Insert Size'''
 This procedure is for checking the insert size of a BioBrick part.  It uses digestion with EcoRI + PstI.
-#Label a 1.5 ml tube for each of the digestions to be set up.
+*Label a 1.5 ml tube for each of the digestions to be set up
-#Transfer about 300 ng into each tube and make up the difference with pure dH2O to 6 ul.  Or just use 6 ul of a miniprep.
+*Transfer about 300 ng into each tube and make up the difference with pure dH2O to 6 ul.  Or just use 6 ul of a miniprep
-#Prepare a master mix as follows:
+*If you have fewer than 4 digestions, add the following to each tube:
+ul dH2O
-#
+ul 2 NEB4 10X buffer
-#
+ul 10X BSA
-#
+.5 ul HF EcoRI
+.5 ul HF PstI
+*If you have 4 or more digestions, prepare a master mix as follows, where N is the number of digestions, then add 14 ul of it to each tube:
+x (N+1) ul dH2O
+x (N+1) ul 2 NEB4 10X buffer
+x (N+1) ul 10X BSA
+.5 x (N+1) ul HF EcoRI
+.5 x (N+1) ul HF PstI
+*Spin down contents of each tube
+*Incubate at 37 C for 10 minutes
+*Add 5 ul blue sample buffer to each and run on gel