User:Tk/Notebook/MF-xfm/2008/04/15

MF-xfm

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Transformation results Many small colonies on Thursday transformations with: outgrowth of only 1 hour with cell density 2,3,4 but not 1 some yeast like contamination on two plates -- probably from the ice water Two colonies on Friday transformations one on the "DNA" plate, a second on the "DNA + BSA" plate not statistically significant Discussion with K. Lee, Krause Lab ref Hedreya93 protocol They concentrate cells 5x (25 ml culture to 5 ml resuspension) They use standard HEPES EPB They wash 3x They electroporate 100 ul cells They add up to 10 ul of DNA (3-5 ug) They elute DNA in water They hold cells on ice with DNA for 10-20 minutes prior to electroporation They resuspend in 900 ul culture The recovery is 1 hour, but sometimes plate directly with little effect on efficiency Phone 706-542-2670, kyungokk@uga.edu Note from Sheppard (UGA) From: Edward Sheppard [1] Sent: Tuesday, April 15, 2008 11:52 AM To: Duncan Krause Subject: Re: FW: Sequence information for pKV104 Duncan Here is the procedure I've been using, it came from Ben. Electroporate Mycoplasma Always run a WT control so that you can see if the transformation worked Label one Gene Pulse cuvette per sample and cool it down in the freezer. From here until 37oC incubation everything is kept on ice. SP-4 No Rx, DNA, Gene Pulse Cuvette, Competent Cell Gene Pulse Cuvette – Biorad 0.2 cm # 165-2086. Combine 5µl Plasmid DNA and competent cells. mix a few times with the pipette. Place them back on Ice. Take your ice bucket containing, SP-4 No Rx, Competent Cells /DNA mix, and cold labeled cuvette to the Gene pulse station. Gene Pulse station Power on Low range Pulse controller set on Low range 100 resistance High Range infinity Capacitance 25 µF To set Gene Pulse II use the raise / lower indicator . set to 2.50 One sample at a time, Pipette cells down side of cuvette. Place in slide loader, ( will only go one direction ) and slide into place. Push both buttons simultaneously and hold for 5 full seconds. Reading should be 4.8 to 5.1. Place 1 ml cold SP-4 No Rx in cuvette on pulsed cells immediately and hold on Ice until all samples are done. Place the cuvette in a disposable 50 ml beaker. 4 cuvette per beaker. Incubate in Mycoplasma incubator for 60 Minutes While Cells are incubation retrieve your PPLO Plates with Appropriate Rx (Gentamicin 18 ug/ml)( Chloramphenacol 24) plates from the refrigerator and dry them in the 37oC incubator. Need 3 per sample. Place in 37oC incubator upside down with the bottoms at an angle on the lids to let the moisture out. Plating Transformated Mycoplasma End of 1-hour incubation retrieve cells and plate on the PPLO Plates with Appropriate Rx (Gentamicin 18 ug/ml)( Chloramphenacol 24) plates you just dried out. Each sample gets 3 plates. The culture coming out of the cuvette is -1 dilution so the first tube is -2. Plate 100 of dilutions -2, -3, -4 . Incubate until you can see colonies and blood overlay. Pick 20-30 colonies each samples and grow up in 500 µl. Transfer any remaining Transformed culture to a 1.5 or 2 ml tube and store -80 oC Edward S. Sheppard University Of Georgia Department of Microbiology Riverbend South Building Room 011 706 542 2670 sheppard@uga.edu Transformation experiment today Wash 45 ml cells 3x with new EPB (no glycerol) 45 ml each time Resuspend to 250 ul Take 50 ul, suspend in 900 ul EPB Take 50 ul, resuspend in 450 ul Transform original concentration (40x), 5x, 10x concentrations Transform each 3x once with fastest possible DNA addition once with DNA left on ice 10 minutes once with DNA left on ice 20 minutes Electroporate at 1.5KV, 1 mm gap add cold medium to 2 ml in the cuvette incubate in medium 60 minutes at 30C plate out on tet plates