IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/15

iGEM Project name 1

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dspB Track Plate taken out at 11:15pm no colonies seems like the ligation had failed; therefore, re-do ligation between his dspB and ccdB chlor backbone Ligation Protocol: "Ligation" protocol from Biobrick 5uL vector: 1.5uL his Tube: his + ccdB -> HC Incubate at room temperature (RT) for 20 minutes Transformation of HC Protocol: SOP; used competent cell #2 Changes: use 5uL instead of 1uL of ligation mix Plates in 37C incubator at 3:05pm Rafael's Method PNK Treatment of DNA Ends on primers Primers: dspB fw rxn1, dspB rev rxn1, dspB fw rxn2, dspB rev rxn2 REAGENTS 1 RXN VOLUME (uL) PNK Stock 1 T4 ligase buffer 1 Primer 8 Tubes: dspB fw rxn1 PNK, dspB rev rxn1 PNK, dspB fw rxn2 PNK, dspB rev rxn2 PNK PCR using Rafael's method PCR Master mix Reagent 1x rxn volume (uL) 5x rxn buffer 5 10mM dNTP 1 sdH2O 6.65 Phusion polymerase 0.1 MgCl2 2 DMSO - 5% 1.25 gDNA 3 Total 25 To PCR tubes, add 3uL of rev + fw to each respective tube fw+rev rxn 1 -> tube 1 fw+rev rxn 2 -> tube 2 Tubes: rxn1, rxn2 PCR Cycles: 98C @ 2min Cycle 27x: 98C @ 10 sec 72C @ 30 sec 72C @ 40 sec 72C @ 10 min 10C @ hold Start: 4:08pm End: 5:10pm Restriction Digest Restriction Digest Supermix REAGENTS 1 RXN VOLUME (uL) Buffer 2 2 BSA 0.2 DNA 10 ddH20 5.8 Total 20 Enzymes: Use XbaI and PstI (1uL each) DNA: ccdb-chlor backbone Digest for 2 hours Assembling the final construct Restriction Digest Restriction Digest Supermix REAGENTS 1 RXN VOLUME (uL) Buffer 2 2 BSA 0.2 DNA 10 ddH20 5.8 Total 20 Enzymes: Use EcoRI and SpeI (1uL each) DNA: prefix and dspB NH Digest for 2 hours Gel verification on RD, ccdB Chlor, PCR products Protocol: biobrick "digest" protocol Changes: load 20uL into each well; 10uL for ladder Gel orientation: Gel orientation 1 kb ladder pre dspB 100bp ladder 1 2 ccdB XP Machine conditions: 0.5x TBE buffer, 100V, 60min Results: Gel extraction Using Invitrogen PureLink Quick Gel Extraction Kit (protocol inside kit) ccdB XP gel weight: 0.1161g pre gel weight: 0.1218g Resuspended in 40uL of buffer Nanodrop: [ccdB XP] = 4.6ng/uL [pre] = 7.0ng/uL PCR using Rafael's method Attempt #2 PCR Master mix Reagent 1x rxn volume (uL) 5x rxn buffer 5 10mM dNTP 1 sdH2O 6.65 Phusion polymerase 0.1 MgCl2 2 DMSO - 5% 1.25 gDNA 3 Total 25 To PCR tubes, add 3uL of rev + fw to each respective tube fw+rev rxn 1 -> tube 1 fw+rev rxn 2 -> tube 2 Tubes: rxn1, rxn2 PCR Cycles: 98C @ 2min Cycle 27x: 98C @ 10 sec 72C @ 30 sec 72C @ 40 sec 72C @ 10 min 10C @ hold Start: 9:55pm End: 10:57pm Gel verification PCR products Attempt #2 Protocol: biobrick "digest" protocol Gel orientation: Gel orientation Quick load 100 bp ladder(last year) 2b W Quick load 100bp ladder (this year) 1 2 ccdB XP Machine conditions: 0.5x TBE buffer, 100V, 45min Results: