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

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Re-PCR with His tag off the genome
Protocol: SOP

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)
Gel orientation: Machine conditions: 0.5x TBE buffer, 100V, 45min Results:
 * Protocol: biobrick "digest" protocol

PCR Purification on his-PCR product

 * Elute with 30uL
 * [His] = 23.5ng/uL

Restriction Digest on his PCR Product
Enzymes: Use XbalI & PstI (1uL each) DNA: his PCR product

Ligation
Calculations $$ ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng)$$ $$3 \times \frac{1200}{2400} \times =  ng$$ $$1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = $$
 * Ratio: 3:1
 * Where x = concentration of insert


 * 0.7uL vector: 17uL insert
 * Protocol: "Ligate" protocol from biobrick
 * Ligase: add 1uL; T4 ligase buffer: add 2uL

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[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
Calculations $$ ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng)$$ $$3 \times \frac{1200}{2400} \times =  ng$$ $$1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = $$
 * Ratio: 3:1
 * 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


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