2012_10_12 EXPERIMENT - ELECTROPORATION AND CELL DENSITY EXPERIMENT
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Commentary documenthttps://docs.google.com/document/d/1btKWgYd1GemMa2mBmg-U72bnu2knSyjf_yGqXo8P1nY/edit
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Original spreadsheet: https://docs.google.com/spreadsheet/ccc?key=0AlVxrZi130nMdHU3Tm1NSGN6Vlp2OEd1OXRpaUROQ3c#gid=21
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Competent cell prep:pg 221 on 10/12/2012. Grew 600 mL of Top10 in TB to OD600 = 0.495. 6 flasks with 100 mL each. Fill 4 centrifuge tubes, spin down, and add the rest of the remaining culture on top. Spinning done at 5,000 rpm (more gently than usual) for 5 min, 4oC. Should have done 10 min. Can't tell if I rinsed more than once with glycerol. Probably only once. Resuspend in 1.2 mL 10% glycerol and aliquot 50 uL batches into autoclaved centrifuge tubes. Flash freeze with liquid nitrogen. Filled about 25 - 30% of a storage box, so probably 25 - 30 tubes were made.
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Electroporation:1.2 kV, 1 cm cuvette (used 4 - 6 times but sterilized between), 25 uF, 200 ohms
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Amt of DNA used for all experiments:2 uL of 1 ng/20 uL
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0.1ng
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2.22E+07plasmidsone microgram (µg, 10^-6 grams) of DNA pieces 1000bp long is 1.52 picomoles (pmol, 10^-12 moles) and 1pmole of DNA pieces 1000bp long will weigh 0.66µg. source: http://www.idtdna.com/pages/docs/educational-resources/molecular-facts-and-figures.pdf?sfvrsn=4
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2.60E+08plasmidscalculated using http://www.promega.com/techserv/tools/biomath/calc01.htm
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4.92E-04maximum efficiency (%)1 pmole pGA 3K3 RFP (3511 bp) = 0.66*1000*(3511/1000) ng = 2717.26 ng
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1.09E+06colonies/ug DNA<-- this is good! While 10^10 is possible, Sambrook says it is practical. 26 J. Sambrook, E. F. Fritsch and T.Maniatis, Molecular Cloning: A Laboratory Manual,ColdSpring Harbor Laboratory Press, New York, 1989.
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2012_10_12 5pm: test the electrocompetent Top10 I just made:
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RFP electroporation numberDilutiondescriptionreplicate numberuL cells usedvolume after dilutionunits10% glycerol to adduL# of colonies counted 12/14:Time cT
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1050 uL of undiluted15050uL0uL984.42
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2050 uL of undiluted25050uL0uL1094.52
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311:1 dilution150100uL50uL324.62
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411:1 dilution250100uL50uL254.88
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551:5 dilution: add 200 uL 150250uL200uL65.08
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651:5 dilution: add 200 uL250250uL200uL05.12
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751:5 dilution: add 200 uL350250uL200uL25.12Concentration factor:
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8101:10 dilution: add 450 uL150500uL450uL05.12500
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9101:10 dilution: add 450 uL250500uL450uL05.18
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10101:10 dilution: add 450 uL350500uL450uL15.2
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111001:1000 dilution1505000uL4950uLInstead dilute some of the 1:10 dilution by 10x: add 50 uL to 450 uL glycerol water05.24
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121001:1000 dilution2505000uL4950uL05.28
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131001:1000 dilution3505000uL4950uL05.24
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% strength# of coloniesSDDilution# of coloniesSD
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100103.57.801047.8
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5028.54.91294.9
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202.73.1433.1
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100.30.6900.6
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10.00.09900.0
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Dilutionlog(# of colonies)
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02.01494035
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11.45484486
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40.4259687323
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9-0.4771212547
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99#NUM!
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cell # reduced but constant volume
constant cell count, volume varying