Hi
I pTZ57R/T Fermentas vector to clone genes in the bacterium E. coli was used.Now I can not do of a single colony obtained plasmid extraction. I have 16 hours in a single colony was grown labial containing antibiotic ampicillin.The problem I have is that the growth of bacteria after 16, even 20 times less. and Plasmid extraction time while adding a solution 2, they are not Lysis and can not be derived plasmid.
Please check, if your antibiotic is working well to make sure that only transformed cells grows on your plate.
Overnight incubation in Ampicillin containing broth LB; is optimum for plasmid isolation. I am following this protocol and successfully isolating the plasmid from the DH5alpha cells.
To 2ml LB Broth + antibiotic inoculate single colony of transformed cells.
Incubate overnight in shaker at 37 ºC.
Pellet down the cells by centrifugation at 6000rpm for 5 min at 4 ºC.
Discard supernatant. Add ice cold 100µl of solution I to the pellet. Vortex to
disperse the cell pellet.
Add 200µl sol. II (freshly prepared) at Room Temperature (RT). Mix very slowly.
Keep on ice for 5 minutes.
Add 150µl of ice cold sol. III. Keep on ice for 3 minutes.
Centrifuge at Maxm speed for 5 min. at 4 ºC.
Collect supernatant (add 1 µl of RNAse, incubate for 1hr on water bath at 37 degree c).
Add 400 µl of 100% ethanol.
Mix gently. Keep on ice for 2 mins.
Centrifuge at Maxm speed for 5 min at 4 ºC.
Discard supernatant. Add 1ml 70% ethanol, disperse pellet. Centrifuge to get pellet.
Discard supernatant, add 50 µl of TE buffer Ph 8. Store at -20 ºC.
use alkali lysis method and various protocol given in sambrook manual. book have 3-4 modified protocol try them u will certainly get results.....
Hi
What I can grasp out of your query is that you have been not able to extract plasmid from a single slowly growing colony on a Ampilicin plate (screen for your clone).
Answer:
1). First of all, the slowly growing single colony, may be an yeast colony, that why your solution 2 was in effective in lysis step.
2) If you are new to plasmid extraction technique. Please start with plasmid extraction from with vector plasmid transformed colony. If you are following classical alkaline lysis method and If you have apprehension that you are screwing up then you a plasmid extraction kit to rule out technical error .
3). Also check the competence of you bacterial cell with standard pUC19 .
I hope this was useful.
Rajkumar.
Hi
alkali lysis method I used, but there was no result. All cases you have mentioned on pBI121 containing bacteria and test the result. But the bacteria containing the pTZ57R / T does not work and does not lyse bacteria and plasmid DNA is extracted.
Hey Zahra!
Did you check whether the plasmid is high or low copy? Did you adjust the culture volume for the plasmid purification ?
The other way around, why do you actually think that your bacteria are not lysed? Which is your observation where you derive this statement from? It is just that I cannot imagine why the lysis may have failed, but if you simply do not retrieve any plasmid, I would not assume a lysis failure in first place (if it is not a contamination as pointed out by Rajkumar).
Best regards!
Christian
As pointed by Christian, sometimes when plasmid copy is low you cant observe the pellet. So, you may need to increase the culture volume for the plasmid purification.
Further, you can run the gel with high volume, you can atleast get bands. You can also concentrate the final plasmid pellet obtained, by dissolving in low volume of TE buffer.
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