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

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QS Track

 * Restriction Digest pCN33 (staph-eco vector), 30, 31, 32, 41, 42, and 43. Plasmids were made in middle of august.
 * Goal is to switch the inserts from PsB1C3, to pCN33.
 * No easy way of telling which colonies actually contain pCN33 with proper inserts.
 * Split the insert and vectors on a gel and then gel extract desired vectors and inserts.


 * Protocol: biobrick digestion (Summary)


 * *amount varies depending on plasmid solution volume needed (see below)
 * Add: 10 uL of pcN33, and 10 uL of 30, 10 uL of 31, 30 uL of 32, 30 uL of 41, 10uL of 42, and 5 uL of 43. Goal is to add 3-5 ug of pDNA.
 * Enzymes: EcoRI and PstI (1uL each to each tube)


 * Put 25 uL of digested mixture onto each lane 0.5X TBE, 1.3% agarose gel. Therefore, need 2 lanes for each type of plasmid digested.
 * Run at 100V, 50 min.


 * Bands were resolved nicely and were bright under UV light. Excised the desired bands. Used Invitrogen kit to extract DNA.
 * No DNA was extracted. Very high absorbance at 220 nm (organic compound contamination).

Restriction Digest

 * Enzymes: Use EcoRI and SpeI (1uL each)
 * DNA: RFP chloraphenicol backbone, and ccdB chloraphenicol backbone
 * Digest for 2 hours at 37C

Gel verification on RD
Gel orientation: Machine conditions: 0.5x TBE buffer, 100V, 60min Results:
 * Protocol: biobrick "digest" protocol
 * Changes: load 20uL into each well; 10uL for ladder

Gel extraction
ccdB chlor gel weight: 0.1999g RFP chlor gel weight: 0.1479g
 * Using Invitrogen PureLink Quick Gel Extraction Kit (protocol inside kit)

Nanodrop:
 * Used 10uL of elution buffer
 * [ccdB chlor] = 18.1ng/uL
 * [RFP chlor] = 21.8ng/uL

Ligation

 * Protocol: biobrick method (number indicates tube number)

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


 * Changes:
 * Total volume: 10uL
 * Incubate at room temperature for 20 minutes
 * Put into -20C immediately after


 * 3uL vector: 1.5uL his/no-his
 * His + RFP chlor -> HR
 * His + ccdB chlor -> HC
 * no his + RFP chlor -> NR
 * no his + ccdB chlor -> NC


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