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pUC19 DNA Storage Buffer Control DNA is supplied in TE Buffer [10 mM Tris-HCl pH 8.0, 0.1 mM EDTA]. Notes on Ligation Reactions

JM109 Competent Cells

Cat. No. M208CC85 Size 1mL Amount 5 x 200 µL pUC19 control

(10 ng/µL)

Ligation reactions inhibit transformation. Less transformants are observed from ligation reactions than from transformations with plasmid DNA. Use 0.5 µL of a ligation reaction per 50 µL of competent cells. For best results, either purify the ligation mixture by ethanol precipitation prior to transformation or dilute the ligation reaction 3-fold in TE buffer and use 1 µL per 50 µL competent cells. Advance Preparations Equilibrate a non-shaking water bath to 42° C. Place SOC Medium at room temperature. Prepare LB agar plates with the appropriate antibiotic. If blue/white screening is desired, the plates should include 40 µg/mL X-gal and 1 mM IPTG. Agar plates should be placed in a 37° incubator C for about 30 min. prior to plating. Transformation Protocol for Chemically Competent JM109 Cells 1. Remove competent cells from -70° and place C directly in ice. Thaw cells for 5 to 10 min. 2. Gently mix cells by tapping tube. 3. Add 1-50 ng of DNA [or 1 µL control DNA] into 50 µL competent cells. Swirl the pipettor tip through the cells while dispensing DNA. Gently tap tube to mix. 4. Place the tubes on ice for 30 min. 5. Heat-shock the cells for 45 sec. in a 42° water C bath. Do not shake. 6. Add 450 µL of room temperature SOC medium to each transformation reaction. 7. Incubate at 37° for one hour, with shaking (225 to C 250 rpm). 8. Spread on LB agar plates containing appropriate antibiotic (e.g., 100 µg/mL ampicillin for control pUC19). 9. Incubate the plates at 37° overnight (12 to 16 C hours).

1 x 10 µL

Store at -70° FOR RESEARCH USE ONLY C. General Description JM109 Competent Cells are chemically competent E.coli cells that are transformed by heat-shock methods. JM109, a K strain bacterium, is recA- and endA- to minimize recombination and improve the quality of plasmid DNA. In addition, the cells carry the F episome which allows blue/white screening and single-stranded DNA rescue. JM109 Competent Cells are suitable for many molecular biology applications, including routine subcloning and cloning in M13 or phagemid systems. Blue/white screening for recombinants requires X-gal and IPTG to be added to the agar plates. The JM109 strain is sensitive to all common antibiotics. General Handling Competent cells are very sensitive to any change in temperature. Cells must be thawed on ice. The transformation should be started immediately after the cells are thawed. Competent cells must be treated gently. Mix cells by swirling or gently tapping the reaction tube. Do not mix by pipetting or vortexing. Once thawed, the cells should be used. Re-freezing thawed competent cells will result in a significant drop in transformation efficiency. Genotype F (traD36, proAB+ lacI , lacZM15) endA1 recA1 hsdR17(rk -, mk ) mcrA supE44 gyrA96 relA1 (lacproAB)

+

q

Efficiency 10 transformants/µg pUC19 DNA

8

M208CC85 July 2007

SOC Medium Formulation 2% Tryptone, 0.5% Yeast Extract, 0.4% glucose, 10 mM NaCl, 2.5 mM KCl, 5 mM MgCl2, 5 mM MgSO4. Related Products Description BL21 Competent Cells BL21 (DE3) Competent Cells BL21 (DE3)pLysS Competent Cells BL21 (DE3)pLysE Competent Cells Value Efficiency AB5TM Competent Cells High Efficiency AB5TM Competent Cells High Efficiency AB10TM Competent Cells ThunderboltTM AB10 ElectroCompetent Cells HB101 Competent Cells SOC Medium X-gal IPTG Rapid Ligation Kit TransfectTM Transfection Reagent Cat. No. M211CC84 M211CC85 M212CC85 M212CC84 M213CC01 M214CC85 M214CC84 M215CC01 M216CC85 M216CC84 M203CC89 M201CC85 M200CC84 M202CC01 M205CC85 M204CC84 M209CC79 M209CC75 M207CC78 M250BM50 M073BC50 M074BC80 M070KT40 M300TF30 Size 20 x 50 µL 5 x 200 µL 5 x 200 µL 20 x 50 µL 96 x 20 µL plate 5 x 200 µL 20 x 50 µL 96 x 20 µL plate 5 x 200 µL 20 x 50 µL 10 x 200 µL 5 x 200 µL 20 x 50 µL 96 x 20 µL plate 5 x 200 µL 20 x 50 µL 5 x 80 µL 5 x 100 µL 10 x 50 µL 10 x 10 mL 1 gram 5 grams 50 reactions 2 x 1.0 mL

Quality Control Cells must have a transformation efficiency of 8 1.0 x 10 transformants/µg pUC19 DNA (nonsaturating conditions).

2222 Michelson Drive Suite 615 Irvine, CA 92612 T: +1-949-226-8094 F: +1-949-608-1975 www.alliancebio.com For Technical service: [email protected]

FOR RESEARCH USE ONLY

M208CC85 July 2007

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