Dear friends,
I am cloning a gene in DH5a cells using pGEX-6P-1 vector. I did double digestion of both gene and vector with EcoR1 and Xho1 with fastdigest enzymes. I got the colonies after transformation and i found desired insert bands in gel after colony PCR. I am Facing following Problems;
1. After picking single colony and growing into L.B broth i extracted plasmid with TIANGEN mini Plasmid Kit . After extraction i lost my gene and vector in the extracted plasmid. After extraction i run 5ul plasmid in the gel but i the gel found empty. There was no vector and gene bands. I did double digestion of extracted plasmid but there was nothing found in the gel. I lost vector and gene after plasmid extraction. I have done this 10 times by checking all the steps but could not find the problem. Kindly help me by sharing your experience.
2. The 2nd problem is that when i transform the gene in DH5a i used Ampicilline in the L.B plates. It grown well and found good colonies . After single colony picking i used L.B broth. I used Ampicilline in broth, the bacteria did not grow with antibiotic. Without antibiotic it grows well. The vector pGEX-6P-1 is ampicilline resistant. Kindly help me to solve this problem.
Looking forward for your valuable answers
Thanks
Ishfaq
What do you do for confirming cloning?
Your second problem shows that you failed in cloning if preparation of your medium be corrected, such as antibiotic concentration in LB broth.
I do colony PCR and then go for plasmid extraction for sequencing but in plasmid extraction i lost gene and vector. extracted liquid is empty. No gene no vector. I dont know either problem is in cloning or something else.
There are different things you could try
1: Pick multiple colonies at least 5 of them and before doing mini prep use cracking buffer to confirm that your plasmid has been subcloned in the broth.
2: Was this your first attempt? Have you tried to transform your plasmid again? There could be something wrong with your mini prep procedure
3: What concentration of Ampicillin are you using? Is it freshly prepared?
Ahmad Jawad Sabir,
I picked multiple colonies and i have done this almost 8 times. I have checked each and every step of plasmid extraction by mini kit but couldn't find the solution. I think there is problem with cloning. Gene maybe not ligated and may not cloned.
But i have spent much time on it but couldn't find the solution of this problem.
What you suggest for this? Maybe contamination issue.
Dear Hafiz,
Colony PCR might give you false positive results as well and it's not trustable every time, There are chances that your insert has not been ligated
Good Luck
Dear Hafiz,
Your problems look very tricky. Did you use appropriate controls in your experiments? It is good to use controls to track the problems. Have you transformed your vector pGEX-6P-1 without your insert? Did you observe the same problem in that case? How old was your LB agar plates? Where did you store your LB agar plates prior to innoculation of your broth? Have you run agarose gel for your ligation products? How did you purify your ligation products? Did you use ethanol precipitation for purification of your ligation products?
You need to revise all the steps to track your problems. Good luck!
Dear Alemu Tekewe,
Thank you for your cooperation by adding your answer regarding my question.
I used the control and the control grows well with antibiotic on LB plates. I prepare fresh LB plates each time and LB agar was stored at 4C refrigerator. I did not run the gel for ligtion product prior transformation. i am purifying the plasmid from bacterial culture by using TIANGEN mini plasmid kit. But after plasmid extraction i lost both gene and vector in extracted product. I dont know where is problem. I think maybe contamination problem. Maybe vector or gene contaminated. Whats your suggestions?
Have you used Gel extraction kit for purification of your cut plasmid and your cut insert? what are the compositions of your ligation reaction? How much was the vector: insert molar ratio/mass ratio for ligation reaction? Did you purify the ligation reaction mixture prior to transformation? or Did you transform non-purified ligation reaction products? Have you used ethanol precipitation for purification of your ligation reaction products? You need to think of my questions to track the problems.
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