Hello,
I want to combine my 1692 bp chitinase gene region with the pBlueScript II SK(+) cloning vector. I added BamHI and HindIII cut sites to the ends of my primers to amplify my gene region. I cut and purify my pcr product. Likewise, I cut and purify my vector. I then perform a 3:1 ligation and transform into E.coli Dh5alpha competent cells. I inoculate on LB medium containing 100 uq/ml amp. I also add X-gal and IPTG to my medium for blue-white colony selection. After 1 day, I choose from white colonies and do both colony PCR and plasmid DNA isolation. Faint bands appear in the gel as a result of colony PCR. In the spectrophotometer measurements, my plasmid DNA isolation results look good, but I cannot see my plasmid in the gel. Apart from this, I also performed the operations using Thermo CloneJET PCR Cloning Kit, but I cannot obtain my recombinant vector.
What could be the problem?
Did you add 3 extra bases at the ends of your primers to ensure good restriction digestion or does the cut site exist exactly at the end of the primers?
When you pcr to test for your inserted product are you using insert specific primers or are you amplifying from the vector into the insert. If your primers are both in the insert then carry over of pcr product will amplify even when the ligation has failed and there is no inserted product
Dear Paul Rutland
First of all thanks for your answer. Yes, I added a 3 bp extension region after the cut region to the tip of my primers. I tried the enzymes on another sample. Both are working.
If there is an error in the ligation, why can my bacteria that do not contain vector inserts give white colonies in AMP medium after transformation?
First ligate your target gene into T-Vector pMD™19 (Simple). Then cut pBlueScript II SK(+) cloning vector with the selected restriction enzymes and then ligate. Hopefully it may work
Dear Rajwali Khan
Thanks for your answer. I don't have the T-Vector pMD™19 vector, but I tried it with the linearized p.JET 1.2/Blunt vector, which is also a PCR cloning vector. As a result, I do not need to choose a blue-white colony. If the insert does not go into the vector, it dies directly. When I seed it on medium containing only AMP, there are colonies but no plasmids in the colonies.
Then i will recommend In-Fusion cloning protocol of Takara Bio. It allow ligation-independent cloning of PCR products into any vector, at any site of linearization. You need to design specific Primers at (https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools) and then follow the protocol of the In-Fusion cloning and ligation kit. I have used it, its easy and time saving protocol.
Good luck
How much plasmid are you loading into your gel? You could always try loading more.
Also, I'm assuming that you grew overnight broth cultures for your plasmid prep with the ampicillin added. If you didn't add the antibiotic, then you'll select for growth of cells that drop the plasmid.
Hi Ahmet Can ,
2 possibilities are that the Amp is getting old and selection is not strong or that the insert is toxic to cells ( try incubating at 25c may help).
The troubleshooting guide from NEB at
https://www.neb.com/tools-and-resources/troubleshooting-guides/troubleshooting-guide-for-cloning
suggests some good control samples to run to assist troubleshooting
may be of interest.
Assuming by faint bands from your colony PCR you mean that you got the right band. If that is the case then the most likely issue is with your plasmid purification. I've worked with cloned chitinases before and there should not be any problem with cloning or expressing them. Did you try a control plasmid to see how that mini prep works for you? Also did the cultures grow well when you grew them and they reached high density? Using a control strain grown alongside you can determine whether the issue was growth of those clones (due to media, cells, phage contamination, ampicillin etc) or a problem with the plasmid preparation.
Katie A S Burnette
Dear Katie,
Thanks for ur answer. I load an average of 300 ng of plasmids into my gel. I tried more, but the result did not change. Since plasmid with high copy number (pBSII) is used in plasmid isolation, I add 100 uq/ml AMP to 1-5 ml LB medium and grow for 16 hours.
Dear Paul Rutland
Thinking that AMP is old, I prepared a new stock AMP and tried it. Again nothing changed. Thank you for the information about the toxic, I will definitely try it. I will also review the link you sent. Thank you so much.
Can I ask one more question ? Where do I need to store the AMP?
Dear Michael J. Benedik
I do this by taking a single colony from petri dishes grown after uncut plasmid transformation as a control during plasmid isolation. I also isolated plasmid DNA from blue colonies. The bands come out clearly. So I don't think I have a problem during plasmid isolation. Additionally, I added gel image after plasmid isolation.
Hi Ahmet,
You can store amp plates at 4c up to 3 weeks and stock solutions at 2-8c for 3 weeks but for long term storage stock solutions keep for at least a yearsat -20c. At 37c amp is stable for 3 days. As a powder amp is very stable .
Hi Ahmet Can , I currently facing the same problem as yours. can you share the solution you got? thanks in advance!
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