I am facing difficulties in cloning a 2.5kb DNA fragment into a vector (pGreen0029). My plates were designed as +ive = pGreen0029, -ive = competent cells (cc), L1-3 = ligation reactions performed on different days.
-*Double digestion of pGreen0029 and pJIT163 with KpnI + XhoI
-Got expected bands and gel purified
-*Ligation at 16, 18 and 22 degree overnight.
*No heat inactivation
Next day transformation
-10 minutes thaw at 4 degree
-4ul ligation rxn per 20 ul cc
-35 minutes on ice
-45 seconds of heat shock at 42 degree
-2-5 mins on ice
-added 300 ul LB media
-1 hour of shaking incubation at 37 degree
-Spreading over LB-agar plate containing 50 ug/ul of kanamycin as selection marker
-16-17 hours of incubation at 37 degree
As expected, getting reasonable colonies on +ive (pGreen0029) plate and no colonies over -ive plate. But not getting colonies over the test plates?
First, I suspected my vector:DNA ratio was abnormal, I fixed the ratio but got same results. Now, I am suspecting my T4 DNA ligase/ T4 DNA ligase buffer have been expired.
When I thawed T4 DNA ligase Buffer it showed white precipitates at bottom. At first, the precipitates got dissolved and then second time, after some days, the T4 DNA ligase buffer showed same white precipitates and precipitates did not dissolve.
What do you suggest about both T4 DNA ligase and buffer? Or any other, like does 2.5kb fragment need any special precaution or strategy for cloning?
Hey Numan Mughal,
A few doubts/suggestions that might help:
1. Why are you seeing colonies in the pGreen0029 plate when there is no insert? No CIP digestion performed? (We do see a few colonies but not many, and why is this not seen in the test plate as well but only in vector alone control?).
2. What V:I molar ratios have you used for the ligation setup? (1:3 V:I should work well).
3. It is quite common that you see precipitates in buffers containing DTT, in such cases vortex or mix the buffer until the precipitates are dissolved. Also, it is important to make sure that the buffer is not freeze-thawed often, since it contains ATP and is prone to degradation. (Aliquotting the buffer into smaller volumes should help).
4. 2.5kb fragments don't require any special precautions usually.
5. What is the source of T4DNA Ligase?. Along with the product they would have given a set of control vector and insert samples. If so use this to rule out the T4DNA Ligase problem.
6. Last but not least, check the efficiency of the competent cells as well, 20ul of comp cells seems very less to me (Unless they are commercial comp cells).
Good luck!
Dear Venkat Mudiyam and Ghulam Abbas ,
Thank you for your suggestions.
1- I am using pGreen0029 as a +ive control (contains kanamycin resistance gene) that's why +ive showing colonies.
2- Yes, I am using 1:3 V:I ratio.
3- I think, there have been many thaw-freeze events, so thinking that new T4 DNA Ligase would work well.
5- I have done transformation successfully many times with 20ul CC but it would be worth trying 50ul CC for transformation. Thank you again for suggestions.
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