IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/17

iGEM Project name 1

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dspB Track Re-PCR with His tag off the genome Protocol: SOP PCR Master mix Reagent 1x rxn volume (uL) 5x rxn buffer 5 10mM dNTP 0.5 sdH2O 9.15 Phusion polymerase 0.1 MgCl2 2 DMSO - 5% 1.25 gDNA 3 10uM FW primer (upHis) 2 10uM RE primer (dw) 2 Total 25 Tubes: His, W1 (with primers), W2 (without primers) PCR Cycles: 98C @ 30sec Cycle 27x: 98C @ 10 sec 72C @ 30 sec 72C @ 40 sec 72C @ 10 min 10C @ hold Start: 3:18pm End: 4:19pm Gel verification for re-PCR (above) Protocol: biobrick "digest" protocol Gel orientation: Gel orientation His 100bp ladder W1 W2 Machine conditions: 0.5x TBE buffer, 100V, 45min Results: PCR Purification on his-PCR product Elute with 30uL [His] = 23.5ng/uL Restriction Digest on his PCR Product Restriction Digest REAGENTS 1 RXN VOLUME (uL) Buffer 2 2 BSA 0.2 DNA 10 ddH20 5.8 Total 20 Enzymes: Use XbalI & PstI (1uL each) DNA: his PCR product Ligation Ratio: 3:1 Calculations [math]\displaystyle{ ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng) }[/math] [math]\displaystyle{ 3 \times \frac{1200}{2400} \times = ng }[/math] [math]\displaystyle{ 1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = }[/math] Where x = concentration of insert 0.7uL vector: 17uL insert Protocol: "Ligate" protocol from biobrick Ligase: add 1uL; T4 ligase buffer: add 2uL [cont. from yesterday]Gel Extraction of His cut at X/P Weight: 0.1548g Using Invitrogen PureLink Quick Gel Extraction Kit (protocol inside kit) 10uL of NCC (1288.3ng/uL) to digest = 1288ng 12883ng of NCC in 20uL = 644.15ng/uL Assume half of this: 322.08ng/uL For EB buffer, add 25uL Nanodrop: [XP dspB extraction] = 6.9ng/uL (25uL in total) Ligation Ratio: 3:1 Calculations [math]\displaystyle{ ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng) }[/math] [math]\displaystyle{ 3 \times \frac{1200}{2400} \times = ng }[/math] [math]\displaystyle{ 1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = }[/math] Where x = concentration of insert Rafael's suggestion: for pre-gel extract, don't use the [] from the nanodrop (7.0ng/uL). Instead, take half the concentration of what was in the digest, in case the agarose is preventing an accurate reading on the nanodrop. 2uL of vector: 5.2uL of His Protocol: "Ligate" protocol from biobrick Tube: XP dspB gel extract + pre gel extract -> pre-xpdspB lig Incubate at RT for 20min Transformation Use 5uL of ligation mix In 37C incubator at 10:00pm