Hi all, I have picked the colony with pipette and tip the colony directly with the PCR mix. The forward primer and reverse primer that I checked is ok with the melting temp 60 C. Then I transferred the pipette tip to 5 ml of LB medium that contain 100 ug/ml of amp. However, after three hours, I done the PCR and no band is shown, BUT THE 5 ml LB medium that contain 100 ug/ml of amp became cloudy. Just 3 clones did not become cloudy. The other 6 clones became cloudy. What is happening? am my cloning is successful?
Is this related to the dipping of the colony to pcr mix? as I just dip the pcr instead of pipette up and down the solution
Hi there,
Let me get the situation correctly,
You performed PCR using the colony of interest, and I assumed you managed to get the DNA band of the correct size. With this information, you proceed to transfer the same pipette tip to the LB for all 9 replicates. However, only 6 replicates became cloudy.
So my opinions are:
1) What primers are you using?
2) Your cloning should be successful if you managed to get the correct DNA band size after PCR
3) Your LB that were cloudy could be contaminants.
What I would do:
1) Patch all the colonies
2) Check the colonies using PCR to confirm cloning is successful
3) If they are correct, proceed with culturing in LB. Take a new colony (confirmed to have correct insert) when culturing.
Hi . Thank you for yr answer. My expression may not be good. The 5 ml of Lb medium is cloudy and there is something (just like bacteria movement) move when I put the tube under the light. I think this is not a contaminants. As the contaminants cannot not be moved and changed the solution became more cloudy after 3 hrs. I strongly suspect this is bacteria growth Andrew Ting
What I meant by contaminants would be something that you do not desire/not of interest. It could be bacteria, fungi, virus, etc.
In cloning, the bacteria may take up empty plasmid (plasmid without insert) and proceed to grow on/in the media (as the plasmid contains ampR gene).
And I also would assume you cultured your colonies on amp plate. So, what I would suggest is to confirm the presence of insert in your colonies, before proceeding. Then culture them in LB.
Have you verified your primers? Many a times there is a problem with primer designing.
Or there might be a contamination.
I suggest that you go for a staining.
Also, plate the clones in a ampicillin LA plate. Then again check the clones with pcr.
I used to maintain my clones in LA plates and they were all right
As Andrew has suggested: confirm insert in colonies by plasmid prep and sequencing/PCR not by colony PCR. There could be inhibitors of PCR present when you pick the colony. Growth of colonies in Amp media is not an indication that everything has worked 100%
You can as well confirm plasmid insert by plasmid extraction from the cells grown in antibiotics and run the plasmid on a gel. The size of the plasmid will confirm whether the plasmid has the gene of interest or not.
Yes..You can still see growth of the colonies in LB medium since they already grew on the agar with Amp. The negative colony PCR clearly implies there is no insert i.e the Plasmid circularizes without taking the insert, and since the Amp resistant gene is present and functional,the bacteria can till grow in LB medium with Amp.
It's better you screen for other colonies and if not successful,repeat the cloning step by increasing the insert vector ratio,incubation time...example leave overnight at 4 degrees..and if your gene is known to be toxic,grow colonies at a lower temperature say 28 degrees
Also during the PCR cloning, you can add a positive control to make sure that your primers really work at that set temperature
Also during the colony PCR , you can add a positive control to make sure that your primers really work at that setted temperature
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