I have been trying to clone a 441bp fragment into a 5.6kb AAV vector backbone using the restriction sites AgeI & ApaI. I use SURE2 cells for the transformation to prevent the ITR regions from getting deleted. My ligation reaction has previously produced no colonies. Here is an overview of my most recent trouble-shooting transformation endeavor:
The ligation reactions occurred overnight at 16C, and were heat inactivated @ 65C 20min.
1uL of DNA transformed into 25uL of competent cells
1) control: uncut vector in ligase buffer +ligase (10ng)
--> only 6 colonies
2) control: uncut vector in water (~17ng)
--> a plate full of colonies (1000+)
3) control: Puc in water (10ng)
--> no colonies
4) cloning ligation reaction (10ng)
--> one colony
Why does the transformation efficiency decrease so dramatically with the uncut vector control when it is in ligation buffer? I have several more constructs to clone, so I really need to work this out. I have cloned into this vector before using these competent cells and did not encounter this problem.
Any suggestions would be most appreciated! Thank you!
~Valerie
Dear Valerie,
I suppose PUC is a kind negative control with a different selection and that's why you get no colony even in that plate.
Anyway I would try a different ligation buffer tube to check if some kind of contaminant is present in the tube you are using now.
Best
The Puc should not be a negative control since I am plating on amp plates. I have no idea why it did not transform, but my guess is that it has become degraded. Also, I use a fresh tube of ligation buffer every time, because the ATP become inactive with too many freeze thaws.
Thank you for the replies!
Tom, I've used two different batches of competent cells. I was using freshly purchased agilent SURE2 cells from our lab but with the poor colony turnout after my transformations I suspected that perhaps their competency was bad. This last run was done using another lab's cells that they have used recently with great success. I plated half of the SOC/cells (250uL). Since the uncut vector in water transformed with no problem I think that the cells are good, but that there is some contaminant in the reaction affecting the transformation. I'm going to concentrate & purify my ligation & uncut vector+ligase buffer (as a control) using Zymo research's DNA clean concentrators and retransform. I am hopeful that this will remove whatever contaminant or condition is causing the problem. Fingers crossed!
I agree with Tom, as last chance to troubleshoot the problem check lot of all batches of ligase buffer. If they are the same and are not good probably they won't work even for the enzyme. You can also try a mock transformation to verify buffer toxicity.
Hi Alberto,
Wouldn't transformation #1 (uncut vector in ligase buffer and ligase +heat inactivation) be the mock transformation determining buffer toxicity?
Anyway, I brought the plates to a department social and got some feedback from our floor's cloning gurus. They suggested that I skip the heat inactivation of the ligase (65C for 20min). The hypothesis is that during this step, the vector denatures and, due to its self-complementary region and Internal Terminal Repeats, it may be annealing to intself in an inappropriate way.
My current plan of action is to transform uncut vector in ligase+ligase buffer without heating. Hopefully this will work it out the transformation complications.
thanks for the feedback! I'll keep you posted.
Probably avoiding the heat inactivation will help (personally I've never done it). But uncut vector in ligase buffer and ligase +heat inactivation is not a mock transformation because anyway will rely on transformation efficiency for bacteria to grow on the selective media. You should put bacteria on a non selective media (no antibiotic) to check for buffer toxicity.
Fingers crossed and go on!
UPDATE: I re-prepped the AAV vector for ligation, eliminating all heating steps. After ligation and transformation, my plates looked beautiful! My experimental plate had many colonies. Before I transformed, I PCR checked my ligation with primers flanking my insert site (added 1uL of ligation to PCR mix). The uncut vector had no band and the uncut vector+insert reaction created the correct size insert.
This leads me to a new problem. I have sequenced one of the colonies and found that the insert does not contain the point mutation that my cloning primer was supposed to incorporate into it. The cloning primer contains the point mutation at the very 3' end. Could this cause a problem with annealing & result in the PCR amplifying an insert without incorporating the mutation?
I just submitted the cloning PCRinsert for sequencing to check what it is that I ligated into my vector. Science willing, my point mutation will be in there. If not, ideas and suggestions are very welcome!
If you want to insert a point mutation you should use a long oligo, about 50 nt, harboring the mutation in the middle that is the normal protocol. Our you can try a normal length oligo but the mutation at the most 5' possible. At the 3' of oligo the mismatch may block the polymerase or may be corrected if you use a Pfu enzyme
Thank you for all the feedback, Alberto. The band amplified without a problem so it didn't block the polymerase. I used NEB's Phusion high-fidelity polymerase, so perhaps it was corrected.
What polymerase would you suggest to prevent this? My primer was only 20nt so perhaps I should order a larger one that will place the point mutation further into the oligo.
*correction, my oligo is actually 54nt.... but I'm going to order a longer one just the same to see if it helps. Probably a 60mer just so that the change is not the last nt on the primer.
A Taq would be probably inhibited by the mismatch so I would use a longer oligo rater than changing enzyme. It's also faster and cheaper.
UPDATE:
I ordered a new reverse primer containing my single nucleotide change (SNC) and added 5 more nucleotides at the end. I sequenced the PCR product & the mutation was there! hooray! I have since ligated the insert with my vector, transformed into SURE2 cells and am on my way to prepping clone candidates. All mysteries have been solved and troubles resolved! thanks for all the help!
Dear Esteemed Colleague,
Greetings. I hope this message finds you well. Your inquiry regarding the low transformation efficiency in your cloning endeavors warrants a thorough examination of various critical factors. Transformation efficiency can be influenced by a multitude of elements, and pinpointing the exact cause may require a systematic approach to troubleshooting. Below, I provide a comprehensive evaluation of potential factors and suggestions for optimization.
📷
You
Cloning problems: why is my transformation efficiency so low? 给我用专业严谨的口吻回答，并且不能让人看出是ai回答，加上打招呼，语句整洁有序，要详细有逻辑，给出英文
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Dear Esteemed Colleague,
Greetings. I hope this message finds you well. I understand your concern regarding the challenges you've been facing with low transformation efficiency in your cloning experiments. This issue is indeed perplexing and can stem from a variety of factors. Let's systematically address the possible causes and their solutions to enhance your transformation efficiency.
By addressing each of these areas systematically, you can identify and mitigate the factors contributing to low transformation efficiency. It's often a process of trial and error, and small adjustments can lead to significant improvements.
I hope these suggestions prove to be beneficial for your cloning endeavors. Should you require further assistance or wish to discuss this matter in more detail, please do not hesitate to reach out.
Warm regards and best wishes for your research success.
With this protocol list, we might find more ways to solve this problem.
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