I insert the 4kb DNA in to PCC1 plasmid. but I have a problem with transformation into top10 component bacteria
plasmid is resistant to chloramphenicol and I add 0.5mg/ml in Lb media
protocol for transform:
1 microliter of ligation + 20 microliter TOP10 competent, mix, 30 min on ice, 2 min on 42 C (thermoblock), then 2 min on ice, then added 1 ml LB with out antibiotic in 37 C, incubate it for 1 hour and transfer to LB agar + 0.5 microgram/ml antibiotic
but after 24 hours there is no colony
although my controls are ok
after 36 hours there was colonies along with satellite
I extract the plasmid but there wasn't any sharp band of plasmid in 0.8% agar
can you tell me what is the problem??
how did you perform your ligation reaction? Did you purify the ligation product? Did you characterize the ligation product? Did you use chromatographic techniques or ethanol precipitation for purification? if ethanol precipitation, did you air-dry the DNA pellet before resuspending them? Did you use a nuclease-free water to resuspend the DNA pellets?
I am also concerned with the incubation time (2 minutes at 42 degrees). It looks a bit longer. competent cells are weak and my die if they are incubated at 42 degree for long time without nutrient. In addition, the plasmids may also come out from the cytoplasm if the transient pores on the membrane remain opened for long time. I often perform transformation heat-shock for 45 seconds at 42 degrees. It works well for me.
I found out that the PCC1 plsamid transform via transfectionand that why I couldnt tranform it
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