Recent replies to "Problems with Transformation / DH5-alpha / Plasmid DNA ??!!"

Reply from Eva Amsen

Eva Amsen — Mon, 18 Aug 2008 04:04:46 -0000

If the empty vector doesn’t give any colonies either, have you tried using a different E. coli strain, like HB101?

Reply from jaya kanthan

jaya kanthan — Wed, 18 Jun 2008 13:12:05 -0000

now i start using topo ta cloning, i also facing problem in transformation.even in control i didn’t get colonies.

Reply from subhadra halder

subhadra halder — Wed, 05 Mar 2008 06:57:06 -0000

dear raghvendra

the problem be the antibiotic you used,so please do check with the conc. or some other antibiotic

thank you

shubhi
nccs pune

Reply from Joe Russert

Joe Russert — Thu, 31 Jan 2008 18:20:50 -0000

I am also having trouble with my transformations. When I started cloning my initial projects were successful, but recently I have not been able to produce colonies. If any are produced they are empty.

From this forum I have decided to set up a control with uncut vector to check my cells.

Is there any thing else that no background would indicate. I am confident in my ligation set up, if anything I am worried that the vector is not cut properly, but this should lead to high background, right?

By background I mean colonies transformed with empty ligated vector.

I am using highly competent dh5alpha cells and pMES vector, and the Roche rapid DNA ligation kit.

Any input would greatly be appreciated.

Reply from Adrian Popescu

Adrian Popescu — Sat, 26 Jan 2008 17:52:10 -0000

What controls do you use? Because that could be the problem

Reply from Wiep Klaas Smits

Wiep Klaas Smits — Fri, 25 Jan 2008 20:29:02 -0000

I dont know what kind of construct you are making, but consider the scenario in which the construct is toxic to the cell. It will cause reduced growth, and curing of the plasmid. Sometimes this can be circumvented using a plasmid backbone that is maintained at lower copy, or using a strain that maintains plasmids at a lower copy number (such as TG90 for instance).
Good luck!

Reply from Dorothy Clyde

Dorothy Clyde — Fri, 25 Jan 2008 14:41:03 -0000

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!
Dot

Reply from Raghavendra V

Raghavendra V — Wed, 23 Jan 2008 09:58:36 -0000

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!

Reply from Amit Kumar Singh

Amit Kumar Singh — Tue, 22 Jan 2008 22:53:34 -0000

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..

Reply from Raghavendra V

Raghavendra V — Tue, 22 Jan 2008 13:53:09 -0000

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!!

Reply from Amit Kumar Singh

Amit Kumar Singh — Tue, 22 Jan 2008 11:18:10 -0000

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

Reply from Raghavendra V

Raghavendra V — Tue, 22 Jan 2008 08:39:18 -0000

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….

Reply from Vandana sharma

Vandana sharma — Tue, 22 Jan 2008 07:05:25 -0000

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

Reply from Raghavendra V

Raghavendra V — Mon, 21 Jan 2008 13:10:00 -0000

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…!!

Reply from Vandana sharma

Vandana sharma — Fri, 18 Jan 2008 07:44:45 -0000

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

Reply from Amit Kumar Singh

Amit Kumar Singh — Wed, 16 Jan 2008 18:55:53 -0000

Hi Raghavendra,
What controls do you use at each step?
Amit