User:Eric Ma/Notebook/iGEM 2010 Lab Prep/2010/05/13

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(Restriction Enzyme Test)
Current revision (12:30, 14 May 2010) (view source)
(Restriction Enzyme Test)
 
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* Digest for ~1-2 hrs (not so crucial) at room temp.
* Digest for ~1-2 hrs (not so crucial) at room temp.
* Note: Initially wanted only 10 µl of water, but in the end, I calculated for 30 µl instead in the master mix for a total of 300 µl of water. Therefore, the reaction may be a bit dilute, which is why I will let this run while I continue writing and rehearsing the podcast instead. The timing shouldn't be too important.
* Note: Initially wanted only 10 µl of water, but in the end, I calculated for 30 µl instead in the master mix for a total of 300 µl of water. Therefore, the reaction may be a bit dilute, which is why I will let this run while I continue writing and rehearsing the podcast instead. The timing shouldn't be too important.
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* Digest ran overnight on ice after ~2h incubation in 37ºC.
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Restriction Enzyme Test

  • To test restriction enzymes, digest DNA with E+P, X+S, E+S, X+P; in addition to this, do four single cuts.
  • We can tell which enzyme is not working (if it is such) by examining which pairs are not cutting properly.
  • I will cut Jason's miniprepped DNA because it had the most amount of DNA present.
  • 4 double digests + 4 single cuts, for further confirmation.
  • Volumes used:
Reagent Control E X S P ES EP XS XP Master Mix
10X BSA22222222220
10X Buffer 222222222220
EcoRI010001100N/A
XbaI001000011N/A
SpeI000101010N/A
PstI000010101N/A
DNA33333333330
sdW303030303030303030300
Total373838383839393939370
  • Digest for ~1-2 hrs (not so crucial) at room temp.
  • Note: Initially wanted only 10 µl of water, but in the end, I calculated for 30 µl instead in the master mix for a total of 300 µl of water. Therefore, the reaction may be a bit dilute, which is why I will let this run while I continue writing and rehearsing the podcast instead. The timing shouldn't be too important.
  • Digest ran overnight on ice after ~2h incubation in 37ºC.


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