I had tried ligation in two different vectors and with two different conditions. I had transformed the whole ligated product in my E.coli cells of efficiency 10^9. The next day I had found in all the plates there are smears but no colonies. My E.coli cells are not contaminated as I had assured it before hand and they all were working fine since I had previously used those cells for other cloning purpose. Then I decided to change the cells and make a new batch of E. coli ultracompetent cells. After preparing the cells, I wanted to check its efficiency and any sort of contamination. So I had transformed a 100ul of E.coli cells with 5pg of pUC 19 vector. I found the same kind of smears and no indiviual colonies. Please help.
Dear Ditsa,
Please check your antibiotic. May be it is not working properly. And your water bath temperature with thermometer while you go for heat shock.
With best regards.
Yes, Shaifali is right, check your antibiotics. You may also want to reduce the amount of ligation product you transform. Say for instance your total ligation reaction volume is 20 ul you transform with 5 or 10ul. instead.
In addition to trying what Shaifali and Afolake mentioned you can try the following:
Are your agar plates freshly poured or taken from the fridge prior to plating out your transformants?
When agar is fresh or cold, then they tend to be a bit watery on the surface (condensation) so when you plate out your transformation and either spread it with spreaders or glass beads - majority of the time you'll get smearing of your colonies across the plate.
Try drying your plates prior to plating, I would take your plates out of the fridge 30 mins prior to plating and stick them in the 37'C incubator slightly ajar (agar side facing down - see attached pic). Same for if the plates were freshly poured, once set you can dry them slightly in the incubator. This will dry out the surface of your agar so when you plate/spread your transformation mix, it should give you nice colonies. However, be aware that because you have dried the surface of the agar prior to plating, any drops of your mix will be soaked up very quickly. So make sure you spread the mix as soon as you put the drop down - i.e. don't add transformation mix to several plates prior to spreading.
Hope it helps!
Dear kindly use some less amonut of ligated product.
Make freshly prepared plates and also check that your agar is right??
Use another fresh antibiotic vial if you have . All these minor things leads to improper results. Thanks and all the best
dear Ditsa,
i think you should check the antibiotic that your vector is susceptible ! as well as maintain highly contaminated free area while you are working.
Technical issue : when the plate was made usually it is recommended to add the antibiotics when the agar is in mild temprature like 35-25 C. if the antibiotics add at high temperature antibiotic can loss its function
You could include a couple of negative controls: plate the competent cells out on selective media without transforming & also transform using sterile water, rather than the plasmid.
Also, how did you ligate? Could the plasmid re-ligate without the insert?
Hope you've sorted it!
I think u added ur antibiotic when it's too hot. So there is a chance of degrade the antibiotics. So kindly let me know this way u tried or not
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