Dear all,
I had a problem with how to insert the gene into plasmid vetor using TA cloning. I used TA-enhancer cloning kit from Nippon Gene. Insert gene was from PCR amplified product 16ng extracted from agarose gel.
After blue/white colony selection overnight and put the LB plate at 4 degree for around 5 hours, I could see the white colonies. So I picked up several white colonies and performed colony PCR( template DNA: 0.5uL from 10ul of LB medium containing one single colony), but when I checked the band of agarose gel, there was no insert gene. I also included positive control which was supplied with the kit. And I would find the band with insert gene.
I kept these LB plate at 4 degree for one week, as time goes on I found that the colonies from sample plate, all of them turned into blue color. What is the problem and how can I insert the gene into this vector sucessfully? And what is the difference between regular PCR and PCR using rTaq enzyme?
Thank you for your kind help in advance.
Dear Jing Zhao do you get PCR fragment in colonyPCR showing no insert or just no signal? If no insert, considering that your control worked, there would be a problem with your PCR fragment that you want to clone:
-either low concentration (16 ng is quite low. You can combine a few PCRs, purify together, and use a bunch more),
-large size of fragment (less efficient transformation),
-size of fragment normal but in frame with the lacZ peptide (this sometimes happens if you don't have a stop in the ORF the peptide can still work, so positive clones can look blue; you could check if that is the case in the hypothetical vector sequence that you would generate. If it is a problem you can try adding one N to the primer.),
-or purity of fragement (You purified from the gel, so i suppose it should be ok. How do you purify?).
As for the polymerase, i think rTaq is just recombinant Taq so it should be the same as normal Taq, but not 100% sure...
Only you have to be careful to amplify your fragment either with a normal Taq not a proofreading polymerase (proofreading Polymerase generates fragments without overhangs, so they can't be cloned in T overhang vectors), or if you use a proofreading Polymerase you have to include an additional step for adding the A overhang.
Hope it helps! Good luck.
Daniela Liebsch Thank you very much for your response.
I could get the PCR fragment in colony PCR showing at around 150bp. PCR fragment that I want to clone was 80bp, so if there was insert gene, it should be around 230bp.
-As for extracting DNA fragment, I just follow the protocol from MagExtractor -PCR & Gel Clean up-TOYOBO. DNA conc. I extracted from agarose gel was 8ng/uL(dissolved with 20uL DW). As the Nippon Gene suggestion, if I want to insert ~200bp, I should add 20.0～32.0ng. Actually the positive control insert is 600bp, with 16ng, it works very well. Maybe I should increase the conc. to see if it helps.
-I only obtained around 5 blue colonies and 20 white colonies from the sample plate. I checked all of them with colony PCR. But still, there were no difference between the blue and white colonies, only no-insert bands.
-I also have same doubts with the purity of fragment. Because by using MagExtractor Kit, when I measured the DNA conc. by NanoDrop, the A260/280 was always around 2.7~3.0. I consulted with the staff from Toyobo company. They suggested me to wash with EtOH several times to improve the purity. But it could not reach to 2.0. Does it effect the results or how could I increase the purity?
-By using rTaq ploymerase, I just hope the product with dA-tali could contribute to transformation.
Really appreciate your help.
Jing Zhao I see, your product is very small! You are right that is should be working with that concentration, but more might help anyway! Did you try to see the fragment size and concentration on a gel after purification? Sometimes with low concentrations and some carry over from the initial or washing buffers (guanidine HCl/TC from the buffers give this kind of problems with disturbing the 230/260/280 ratios) you get a higher concentration reading than is real. You can get an idea by not only seeing the od260 but by looking at the whole graph if you have a good peak at 260 or some weird signal); In general I find it better to check on gel at least if below 15 ng/ul. It also allows you to see if there are any other bands that aren't supposed to be there. About purity, I don't know how it works with this kit, but more washing and trying to get as much of the buffers out as possible between steps (centrifuging, pietting, whatever applies here?) can indeed help. Maybe you could increase efficiency for dA attachment by add a little more dATP after the last step and incubate like 10 min more? Goof luck!
Daniela Liebsch Regarding the assessment of the fragment size and concetration after purification what you mentioned, so I just run DNA I got by gel eletrophoresis again? In fact, the original product exhibited a single distinct band without any accompanying artifacts or smearing. Here, I also attached a figure depicting the peak during the measurement and based on this graph, the peak appeared around od230.:(
To increase the efficiency for addition A to PCR product, I am planning to incubate at 72 degree for 15mins. Keep my finger cross.
Jing Zhao Yeah, if the peak is mainly at 230 it is the contamination from the buffer. There is almost nothing visible at 260 and it will be affected by the contaminant, so the reading for concentration is not informative and you can't really trust it. It might be that you have very little fragment. That's why I suggest to run about 5 ul (or as much as u can spare) of the 20 ul in which u recovered the purified fragement on gel again to see the actual concentration (in comparison with a ladder with defined amounts in each band).
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