Problems with Transformation / DH5-alpha / Plasmid DNA ??!!
Raghavendra V
Wednesday, 16 January 2008 16:10 UTC
We have cloned a gene, and transformed it into DH5-alpha E.coli cells with suitable antibiotic (of course in the right concentration). The problem is, after incubating O/N at 37 deg C, we notice very tiny colonies on the LB-agar plate, and with great difficulty if we are able to locate, and inoculate a single colony into LB for Miniprep, at the end of the procedure we do not find any plasmid DNA!! (on 1% TAE agarose gel!!) Can any of you give a solution for this problem?!
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Hi Raghavendra,
What controls do you use at each step?
Amit -
I think the problem is with ligation which may have resulted in unwanted combinations of vector:vetor or insert :insert and a very few insert :vector .
You first need to be sure about the quantity and ratio of your vector and insert and then proceed with ligation.
Another thing could be are your vector and plasmid digested with same restriction enzymes or are you using TA cloning.
In first case there may be self ligatio !
So u need to look for that
good luck1 -
Hi AKS and VS, first thanks a lot for you guys for your reply!
Aks, the control varies from step to step…like for eg. during ligation, I set-up a reaction without insert, which serves as control!!
VS, we usualy run 1% TAE agarose gel electrophoresis, quantitate visually, and then set-up ligation accordingly (considering the ration of Plasmid:Vector based on gel)Also, according to you, if at least there is very few Vector:Insert ration…even then at least we need to get quite good colonies…if not many at least?!
Also, I have one more question…is there any way to quantitate Plasmid DNA done by Maxiprep, OTHER THAN Spectrophotometric(OD 260/280)method??!!
Please do comment on the above…!!
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The easiest way to quantitiate plasmid in my opinion is running it on gel only.
I was asking you about the vector :insert ratio because I am also facing the same problem and I think my vector and plasmid are not ligated thats why I end up with a few colonoes1 -
Thanks for the reply VS…I do get colonies, but they will be so tiny that its very difficult to pick a single colony for Minipreps or so…also end up with nothing at the end!!...hmmm…I know running on the gel we can quantitate DNA, but it will not give acurate concentration right?! for futher down-stream processing!!...suggest any alternative…??!!
please do comment….
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Hi Raghavendra,
The colonies you are seeing might just be unspecific colonies because of antibiotic degradation. we always start getting these colonies if you incubate ur plates little long.If you have following controls, you may find out wots wrong..
1. uncut DNA vector (With antibiotic)-
Expected colonies: many ~100 depending upon DNA concentration. (if you find No colonies here, it means ur competent cells are not good, or transformation didn’t work)2. uncut DNA vector (NO antibiotic):
Expected colonies: Millions because of No antibiotic. (if you find No colonies here, it means that ur competent cells are dead)3. No DNA (With antibiotic)
Expected colonies: No colonies (if you find colonies here it means ur antibiotic is not good)4. No DNA (NO antibiotic)
Expected colonies: Millions,5. DNA Ligation (With antibiotic)
6. DNA Ligation (NO antibiotic)
You can use ur ligation controls as well..
I hope this will help..
good luck..
Amit -
Hi AKS, thanks for the reply! Yaar, thats really a good set of solutions…lemme try them out, and get back to you!!
How about estimating Plasmid DNA? (other than by Spec. method!!)
Cheers!!
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OD is fine.. otherwise you can also run ur DNA with any any standard DNA ladder where you can compare the intensity of bands..
*you can include one more control like.. just leave a plate overnight (no cells, only antibiotic), to check any background..
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Hi AKS, thanks for the reply!! Hope you got my point right!!...we are getting lot of colonies, but the problem is they are damn tiny, and we end-up in no DNA at the end of preps!!
Yes, as you said, we can compare the intensity of bands on gel, but we can not estimate the exact quantity right?!...esp. for further downstream processing like for eg.if we need to subject it for ligation!
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I think Amit is right; the tiny colonies you describe tend to appear when you’ve incubated your plates too long and the antibiotic has started to break down. Because the antibiotic selection is no longer effective, the bacteria in these colonies won’t contain the plasmid – which is why you don’t get DNA when you do a mini-prep and run your gel.
So, it seems like your transformation isn’t working; probably either the ligation isn’t working or your cells aren’t competent. Follow Amit’s advice and carry out his suggested controls to work out which.
I wouldn’t worry about the ‘exact’ concentration of the DNA you use for setting up ligations. I always found that estimating the concentration from gels (as suggested by Amit) was more reliable than spec readings! Regardless, you should aim to set up ligations using different ratios of vector:insert; where the amount and concentration of DNA allowed it, I always set up 3:1, 1:1 and 1:3.
You should also bear in mind that not all vectors and inserts are as efficient in ligations as others. In general, large vectors and/or large inserts tend to be more troublesome. Blunt ended fragments also tend to ligate less efficiently. In cases where you know the ligation efficiency is going to be low, you should use the maximum competency cells available to you for transformation.
Hope this helps – and let us know how you get on with Amit’s suggestions.
Good luck!
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