I am trying to ligate 4818 bp vector with 4 different inserts (3kb, 3.5 kb, 5.3kb and 6.5 kb) separately which would yield me 4 different products. the vector and inserts are cut with two different Restriction enzymes (Mlu1 and Xho1). I used 1:3 vector: insert ratio and kept ligation reactions at 4 deg for overnight before transformation but each time i get numerous colonies on all 4 plates. I tested the antibiotic efficiency by plating only competent cells over an agar plate conatining antibiotic and no colony appeared, which means antiobiotic (ampicillin) works fine. Please suggest me what should I do? I am using 0.2 microlitres T4 DNA ligase from Thermo Scientific (EL0012) for ligation.
I assume you have picked a number of these colonies and they don't contain the required insert. Try including a control containing only the recipient vector without insert. If this yields numerous colonies then you will know that non-cut plasmid is present. I generally run the cut recipient vector on a gel for a long time to see if there is any remaining uncut vector, and I always include a control so that I can see what percentage of colonies may be the result of uncut vector.
After transformation, each competent cell having received vector must multiply to give a colony. So, in each plates you obtain numerous colonies: this is the normal outcome. Subsequently, you must distinguish between cells received the empty vector from those received the vector + insert.
Thanx Richard and Basma! Richard you have given the answer to my question which I couldn't find. and Basma I screened 50 colonies of each product. All were either false positives or no band at all in colony pcr.
I have suggsetion for you. you can do gel cracking for some colonies and compare them with empty vector on electrophoresis. another thing, you should be sure about insert and vector concentration. if you take good bands from insert and vector after purification, next step is being sure about your restriction enzyme and ligase that they work correctly or not. last thing!! insert 6.5 kb is toooo long for vector 4.8kb. you must have bigger vector for this insert
I would like to add here few things/protocol pla:
1. Use empty vector as a control
2. Since your combatant bacteria are sensitive to the antibiotic Amp 100ug/ml so your transformation was okay and might have the insert with F-plasmid.
3. If you had used 200-50 ul SOC medium after transformation you can incubate it for only 30 min instead of 1h or dilute the soc medium with 1:3 with LB and use only 50 ul to plate them.
4. Pick some isolated colonies (about 5 Only) and then screen for your insert usin conventional PCR using ONLY 1-2 ul and check if you get your defined band or not (please use the internal primer). Do not forget to use bacteria contain empty vector as a negative control.
Please update all of us if you still have any problem,
Zack
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