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

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dspB Track Gel verification on digestion Protocol: gel verification protocol in Protocol (SOP) Machine conditions: 0.5x TBE buffer, 110V, 60min Gel orientation: Gel orientation D G 100bp ladder suf 1 kb ladder Results: 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 Restriction Digest REAGENTS 1 RXN VOLUME (uL) Buffer 4 2 BSA 0.2 DNA x ddH20 7.8 Total 20 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 Protocol: gel verification protocol in Protocol (SOP) Machine conditions: 0.5x TBE buffer, 110V, 60min Gel orientation: Gel orientation C2 C4 D4 100bp ladder H2 H4 G4 term4 1 kb ladder Results: 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 Restriction Digest REAGENTS 1 RXN VOLUME (uL) Buffer 4 2 BSA 0.2 DNA 8 ddH20 7.8 Total 20 DNA: Cut D and G at E/S Add 1uL of each appropriate enzyme Gel verification on digestion Protocol: gel verification protocol in Protocol (SOP) Machine conditions: 0.5x TBE buffer, 100V, 60min Gel orientation: Gel orientation C D 100bp ladder H G 1 kb ladder Results: [[Image:‎|300px]] No cuts...... PROBLEM: construct is E-X-dspB-P-S-P