Haynes Lab:Notebook/CRISPR Editing/2015/05/16

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05/16/2015

Cloning GFP behind Cas9
Will do ligation before and after purifying with Qiaquick PCR purification kit which will get rid of the 23 basepairs cut out of the backbone and the >10bps cut off the ends of the insert
before purification

50ng backbone (8486bp) = 50ng /(1000ng/50ul) = 2.5ul
10ng insert (824bp) = 10ng / (40ng/25ul) = 6.25ul

mixed backbone and insert, backbone only, brought both to 10ul with water
added 10ul 2x Roche ligation buffer
added 1ul Roche T4 ligase
mixed and spun
incubated at RT on benchtop for 15-20mins
moved 2ul of each reaction to a new tube, added 10ul water
water only control
added 50ul of my DH5αTs or 50ul of KAH's DH5αTs
incubate on ice for 30min
heat shock at 42°C for 35s
incubate on ice for 2min
add 750ul SOC to each tube
shake at 37°C for 1hour
plate all cells

Cell type plasmid 1 plasmid 2 rep 260 280 260/280 ng/µL ul for 400ng
Luc14GFPg03420.0330.0191.79133.35411.99256461
Luc14GFPg03430.0270.0141.95326.81414.91758037
Luc14GFPKAH22820.030.0161.85330.42713.14621882
Luc14GFPKAH22830.0330.0162.09632.5612.28501229
Luc14KAH228g03420.0320.0181.81431.86812.55177608
Luc14KAH228g03430.0320.0171.86931.6412.64222503
Gal4-EED +doxGFPg03420.0330.0171.91633.46611.95242933
Gal4-EED +doxGFPg03430.0270.0141.95326.52115.08238754
Gal4-EED +doxGFPKAH22820.030.0171.7530.46613.12939014
Gal4-EED +doxGFPKAH22830.0230.0121.96422.79417.54847767
Gal4-EED +doxKAH228g03420.0250.0131.93524.57516.27670397
Gal4-EED +doxKAH228g03430.0290.0151.92529.36913.61980319



cell type ug plasmid ul lipo rep 260 280 260/280 ng/µL ul for 400ng
Gal4-EED0.5310.0340.0181.9133.54511.92428082
Gal4-EED0.5320.0310.0171.88631.16512.83491096
Gal4-EED1310.030.0151.93929.99413.33600053
Gal4-EED1320.0310.0181.72130.67413.04035991
Gal4-EED0.5510.0280.0151.94328.4414.06469761
Gal4-EED0.5520.0270.0141.92127.3414.63057791
Gal4-EED1510.030.0161.88430.30313.2000132
Gal4-EED1520.0290.0151.93828.81313.88262243



' Cell type plasmid 1 plasmid 2 rep ng/µL ul for 400ng ul annealing buff ul water sur
1Luc14GFPg034233.35411.992.006.01+
2Luc14GFPg034326.81414.922.003.08+
3Luc14KAH228g034231.86812.552.005.45+
4Luc14KAH228g034331.6412.642.005.36+
5Gal4-EED +doxGFPg034233.46611.952.006.05+
6Gal4-EED +doxGFPg034326.52115.082.002.92+
7Gal4-EED +doxKAH228g034224.57516.282.001.72+
8Gal4-EED +doxKAH228g034329.36913.622.004.38+
9Luc14GFPKAH228230.42713.152.004.85+
10Luc14KAH228g034231.86812.552.005.45-
11Gal4-EED +doxKAH228g034224.57516.282.001.72-


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