Editing Competent-Cells (section)

== <br/>Electroporation Protocol ==

Ligation at 16 oC o/n, 

Add 1 µl of 1 µg/µl yeast tRNA,&nbsp; 50 µl 100% cold ethanol, put into −20°C freezer for 15 min<br/>Spin&nbsp; 10 min<br/>Wash with 100 µl of 70% ethanol<br/>Air-dry<br/>Resuspend&nbsp; in 1 µl of ultrapure water.

Transform with 20 µl electrocompetent cells

<br/>The electroporation protocol will vary depending on the strain so this protocol may need to be optimized. For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.

Turn on electroporator and set to 1.7-2.5 kv (optimize for strain), 200 ohms and 25 µF.<br/>Place recovery SOC in 37°C water bath.<br/>Pre-warm LB-antibiotic plates at 37°C.<br/>Thaw cells on ice for 10 min or use freshly made cells.<br/>Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.<br/>Flick the tube containing cells a few times to mix and add 25 µl to the microcentrifuge tubes.<br/>Add 1 µl of a 10 pg/µl DNA solution (in DI water) to the cells in the microcentrifuge tube.<br/>Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down).<br/>Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.<br/>Rotate in the 37°C incubator for 1 h.<br/>Make appropriate dilutions. When using 10 pg of DNA, make two dilutions:<br/>Dilute 10 µl cells into 990 µl SOC and plate 100 µl. (1000-fold dilution)<br/>Dilute 100 µl cells into 900 µl SOC and plate 100 µl. (100-fold dilution)<br/>Incubate overnight at 37°C.<br/>Calculation:<br/>If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml)

cfu/ µg = (colonies counted*1000) / (0.00001 µg pUC19)

'''Procedure'''

•Inoculate 250 mL SOB media with 10 - 12 colonies from a freshly streaked plate.

•Grow to an OD600 of 0.94 at 18°C (or RT). (Doubling time is 4-10 hours). (1/04: 9.7 hr for INVaF’)

•Chill on ice for about 10 minutes

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*From this point on, it is critical to keep cells below 4°C**

*
*Work in cold room when possible**

•Pellet cells at 2500 x g for 10 minutes at 4°C.

•Resuspend cells gently in 80 mL of ice cold TB.

•Leave on ice for ~10 min.

•Pellet cells as before

•Resuspend cells gently in 10 mL ice cold TB.

•Add 10 mL ice cold TB + 14% DMSO.

•Wait 10 minutes, then aliquot into pre-cooled 1.5 mL tubes, and freeze them in liquid nitrogen.

•Store in -70 oC freezer.





'''Transformation'''

•Thaw cells on ice. (If desired, split comp cells to pre-cooled tubes.&nbsp; Can use&nbsp;12.5 µL/transformation.)

•(Optional for ~2-3X efficiency increase: Add 4 µL of 0.5 Μ (or 0.14 µL of 100%) β-mercaptoethanol to cells per 100 µL competent cells. (20 mΜ final)

•Add DNA (I add <3 µL per 100 µL comp cells)

Incubate in ice for ~'''30''' minutes. (Longer incubations seem to help for plasmids >10 kb.)

•Heat shock at 42°C for '''30''' seconds then place on ice for another 2 minutes.

•Add 0.9 mL pre-warmed SOC media. (LB works but might affect efficiency)

• 37°C for 45 minutes (or longer).

(Some people suggest shaking, others say it kills the cells)

•Plate