Hello Everybody, Hope you doing good.. I am facing the problem in colony PCRof Agrobacterium EHA105, I did it abou20 times but failed every time. Can any body tell me the best way to deal with this. EHaA grow normally on antibiotic plate, giives normal morpholoogy buut when i do colony PCR, itss negative. I use suspension in water, LB (shaking for 4 hour), boiling for 10 miinutes, using fragment primers, using vectpr pprimers.. May be every thing in my mind. I tried to miniprep but my kit is in compatible with this strain. If any one has some idea, please share it, i am dying with wastage of time. Thank you so much. Please share your ideas, PCR protocol or making miniprep, Thank you so much.
Hi....
Since your EHA is growing on selection pressure means pIasmid is present. I think amount of inoculum ur using for pcr is more that's why it is getting inhibited. Generally I will take single colony and inoculate in water and make suspension of it then heat it at 95 for 10 min and cool it at 4 for 2 to 3 min. Brief centrifuge the tube and take super-natent 2 to 3 ul as template for pcr without taking any cell debris. It works very fine.
Use plasmid isolated from e. coli as + ve control to ensure that there is no inhibition of pcr.
Good luck.
You could try just picking the colony on a pipette tip and rinsing it in the pcr mix then put the tip in growth medium in a 96 well plate for future use of the colony if it pcrs positive .. Good primers will often work in this situation but you may want to run 35 or more cycles of pcr and have a positive and negative control dna purified properly to show that the primers can work. raise the magnesium concentration slightly in case the small amount of cell debris interferes with the pcr. some enzymes are more robust than others in the presence of contaminating material so if you are sure that your primers are good you could try using another polymerase if colleagues have a different one spare
I think no need to do miniprep. Just pick part of colony and mix with PCR mixture. Do direct PCR with appropriate primer pairs. I think Paul Rutland gave you a correct suggestion.
Thank you all for your nice suggestions.. I am working on it and hope that i will get the good results soon.. thank you so much..
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