IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/22

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Gel verification on digestion
Gel orientation: Results:
 * Protocol: gel verification protocol in Protocol (SOP)
 * Machine conditions: 0.5x TBE buffer, 110V, 60min


 * DspB isn't cutting. Second try.
 * Next step (troubleshooting): try using the RD buffer that has 100% activity for each enzyme in a reaction (NEbuffer2 = 100% for all of them already, but try using NEbuffer 4)

Re-Restriction Digest

 * Protocol: "digest" protocol from biobrick


 * x = amount of DNA
 * DNA:
 * Cut terminator (suffix) at E/X (use x = 15uL)
 * Cut D and G at E/S (use x = 10uL)
 * Add 1uL of each appropriate enzyme

Tubes: C4, D4, G4, H4, C2, H2, term4
 * 4 = used buffer4; 2 = used buffer2

Gel verification on digestion
Gel orientation: Results:
 * Protocol: gel verification protocol in Protocol (SOP)
 * Machine conditions: 0.5x TBE buffer, 110V, 60min


 * Still single cuts
 * Tried buffer 2 and 4 -> both didn't work
 * SpeI dead?

Re-Restriction Digest with new SpeI

 * Protocol: "digest" protocol from biobrick


 * DNA:
 * Cut D and G at E/S
 * Add 1uL of each appropriate enzyme

Gel verification on digestion
Gel orientation: Results:
 * Protocol: gel verification protocol in Protocol (SOP)
 * Machine conditions: 0.5x TBE buffer, 100V, 60min


 * No cuts......
 * PROBLEM: construct is E-X-dspB-P-S-P


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