DH5 a λ pir competent cells*

DH5 a λ pir competent cells*

Product name: DH5 α λ pir Chemically Competitive Cell
Product Code: G6018-10 (10 pcs 100ul), G6018-100 (100 pcs 100ul)
Specifications/Introduction: G6018-10 (10 units 100ul), G6018-100 (100 units 100ul)

genotype

F- φ80 lac Z△M15△(lacZYA- -arg F) LAMpir U169 endA1

recAl hsdR17(rk- ， mk+) supE44 λ- thi -1 gyrA96 relAl phoA

Briefly explain|

The CLBio DH5a into PIR strain is derived from DH5a, and LAMpir was introduced into the DH5a E. coli genome, which is DH5a into PIR. This strain can express PIR protein, allowing plasmids containing R6Kg ori replicons to replicate normally. The DH5a strain lacks endonuclease A (endA), which increases the yield and quality of plasmid DNA; Recombinase defective type (recA) reduces the probability of homologous recombination of inserted fragments, ensuring the stability of inserted DNA; The presence of lacZOM15 allows DH5a to be used for blue and white spot screening; However, the growth rate of DH5a entering the Pir strain is slow, and the growth time should be extended during plate culture or liquid shaking. CLBio DH5a λ pir competent cells were prepared using a special process, and the pUC19 plasmid detection showed a transformation efficiency of>1X109 cfu/μ g DNA.

Operation Instructions:

1. Take out DH5a λ pir competent cells from -80 ° C and quickly insert them into ice. After 5 minutes, wait for the bacterial mass to melt, add the target DNA (plasmid or ligation product) and mix well by hand at the bottom of the EP tube. Let it stand in ice for 25 minutes.

Heat shock in a 2.42 * C water bath for 45 seconds, quickly return to ice Stand still for 2 minutes, shaking will reduce conversion efficiency.

3. Add 700 μ 1 sterile LB medium without antibiotics to the centrifuge tube, mix well, and resuscitate at 37 ° C and 200 rpm for 70 minutes.

4. Centrifuge at 5000 rpm for 1 minute to collect the bacterial cells. Take about 100 μ l of supernatant and gently blow and coat the heavy bacterial blocks onto LB medium containing the corresponding antibiotics.

5. Invert the plate and place it in a 37 ° C incubator for at least 18 hours.

Precautions：

1. Sensory cellssuggestionSlowly melt in ice. Insert the target DNA into the ice within 8 minutes, and do not leave it in the ice for too long as it will reduce the conversion efficiency.

2. The DH5a λ pir strain grows slowly, and the growth time of the bacterial cells should be extended (at 37 ° C for more than 18 hours) when cultured on plates or shaken in liquid.

3. Converting high concentration plasmids or efficient ligation products can correspondingly reduce the amount of bacteria ultimately used for plate coating.

DH5 a λ pir competent cells*