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

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(Restriction Enzyme Test)
(Restriction Enzyme Test)
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| 10X Buffer 2||2||2||2||2||2||2||2||2||2||20
| 10X Buffer 2||2||2||2||2||2||2||2||2||2||20
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| EcoRI||0||1||0||0||0||1||1||0||0||
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| EcoRI||0||1||0||0||0||1||1||0||0||N/A
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|-
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| XbaI||0||0||1||0||0||0||0||1||1||
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| XbaI||0||0||1||0||0||0||0||1||1||N/A
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| SpeI||0||0||0||1||0||1||0||1||0||
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| SpeI||0||0||0||1||0||1||0||1||0||N/A
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| PstI||0||0||0||0||1||0||1||0||1||
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| PstI||0||0||0||0||1||0||1||0||1||N/A
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| DNA||3||3||3||3||3||3||3||3||3||30
| DNA||3||3||3||3||3||3||3||3||3||30

Revision as of 18:43, 13 May 2010

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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.



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