If you can find a restriction enzyme which cuts in your vecctor but not insert. Digest the ligation mix with the enzyme and then transform. This will remove the background of vector religation. Also with pBSK you can go for blue white screening

A couple of suggestions are:

A. Use freshly cut vector/insert - Purification post digestion is not absolutely essential.

B. Work with different ligation ratios. Vector:Insert - 1:5, 1:10, 1:20.

C. Perform an overnight ligation at 16 degrees.

D. As recommended by Vanita, use blue white screening to scan your true clone

E. If you are still facing difficulties, or are short of time, use gateway cloning.

hello Vanita

Thank u for your suggestion ill try with it

Hai Deepak

I used the same procedure wat u mentioned but the thing is negative contol i.e CIP treated vector also ligating, i could able to huge no of colonies on negative plate

Dear Vanita

If i cut the ligated mixture with the enzyme which is present in vector but not in the insert as u mentioned, itll cleave the vector which is having my insert, then how it'll be transformed into cell, if digested vector couldnt get into cell means how can my gene of intrest containing vector 'll get in after digested with another RE

Dear Suloka,

Are you performing cloning using a single enzyme digest, eg. using a single enzyme site such as EcoRI, BamHI etc? Or are you using a combination of two enzymes, such as EcoRI+BamHI, etc?

The latter would be better.

One of the main sources of 'religated' vector is actually vector that was never digested. Did you run the digested vector on a gel and purify band to make sure no undigested vector was carried over into the ligation mix? Run a sample of undigested vector alongside to identify the correct band.

By the way, the method Vanita talks about is where cloning the insert into the vector destroys a restriction site in the original vector so that only non-insert-containing vector will be digested.

Hi Suloka,

Sorry I was not clearer earlier. What I meant was if your doing a directional cloning cut with an enzyme which is in MCS of your vector which is being removed during cloning or if your doing a blunt end then cut with the enzyme whose site you are using for inserting the insert.

Hi

CIP is a very active enzyme and small amounts of it have inhibitory effect on ligation reaction. You may CIP treat vector for 1 hour at 37 degrees and then inactive the enzyme for 1 hour at 65 degrees. you'd better to recover the vector band from agarose gel. you may no need to kinase treat the insert. For the background problem you may check the efficiency of antibiotic. You may have a control ligation reaction comprises of vector+buffer+water+ligase (without insert) in all cloning experiments. It helps you to judge the ligation reaction.

God speed you

If you are trying to clone PCR fragments, you are better off buying pGEM-T which is prec-cut vector with a T overhang. You can also use blue/white selection with this vector. The success rate is much higher than trying to clone into T-tailed bluescript.

Hai deepak,

Its single enzyme sma1 ( blunt end ) digestion

hai Vanitha

I got the idea now. I'm using blunt end digest with sma 1 as you say i wanna digest the ligation mixture with sma1 again, then self ligated one will digest and ligated wil transfered into cell.

One more thing if i treat the ligation mix again with an enzyme, how could i remove the enzme from the mixture i have to purifi the ligation mix r what..?

Hi

Firstly you may inactive the enzyme (by heat), then add absolute ethanol (2 volumes), centrifuge at max speed for 10 min, add 1 ml ethanol 70%, centrifuge again and dissolve DNA in ddH2O.

God speed you

Hi,

Single enzyme blunt end cloning are quite tricky. And I am assuming that Sma1 is a unique enzyme within the vector, which makes the case more complicated. You will not be able to use Vanitha's strategy here - because cutting the ligated mixture using Sma1 will also get the insert out. It would have worked good, when you were cloning between two sites in the vector, and you could then make a cut between the two sites in the ligation mix to remove the religated vector.

If you can change your cloning strategy a bit by employing another site in addition to Sma1, it should work out easier for you. Regardless if you are continuing with the current strategy, a couple of recommendations that I can make are:

A. Use a fresh digested vector. Run it on the gel next to the undigested vector and confirm that the vector is completely digested.

B. Treat the digested, purified vector by CIP.

C. Digest your inserts, confirm their digest on the gel - perform kinase treatment.

D. Use a mixture of ligation ratios, by increasing the insert amount. Keep the following controls for verification - Digested vector alone+/-ligase & Digested vector treated with CIP alone+/-ligase.

E. Perform a blue/white screen for efficient screening.

All the best!

In addition to D. Iyer's post, a few more tips:

- use PEG 8000 solution, which is supplied with some ligases

- ligate ON at 8-16°C if you have difficult ligations (= anything else than short fragments with sticky ends on both sides)

- Aliquot your ligase buffer to avoid freeze-thawe cycles which will pretty quickly destroy it.

Some more information can be found here: https://www.neb.com/tools-and-resources/usage-guidelines/tips-for-maximizing-ligation-efficiencies.

Hi Suloka,

Unless the insert and vector site have a sequence which will rebuilt the site u can use SmaI.

If you want u can heat treat the mix to remove the enzyme but I generally transform the mix directly. I have used the strategy for cloning at SmaI site so this is from experience.

Just check that your vector and insert ends don't remake the site.

Also use large insert vector ratios. I have used up to 20:1

all the best.

Dear Vanita,

hw to check the insert vector is remaking the digested site r what..?

And as I;m using blunt end if i digest the insert it'll creat overhangs which can wont fit for blunt site, thats the reason i used Pfu polymease to obtain the blunt end pcr product.

And i'm using PEG 6000 not PEG 8000, Does it make scence..? I'm using thermoscientic enzymes not NEB

Hi Suloka,

The SmaI site is 5'-CCCGGG-3' and if you digest the vector the ends would be 5'CCC-3'. Just check your primer so that it doesn't have 5'-GGG-3'. otherwise the restriction site would reconstitute on blunt end ligation.

About PEG use the solution which is provided with the ligase. The brand of enzymes should not make a difference.

Please see if the vector is completely digested before using it for ligation...if there are undigested plasmid DNA bands...they will give high background colonies...try to gel elute the single digested band of vector DNA as well...

hai Vanita,

I tried your method i got one clone too, but i'm tryiing to double digest it, to release my fragment out but its not giving any fragments out. what shall i do....?

Well Suloka

N things could have got wrong

Are you confirming with PCR. If so then you might want to optimize the digestion. A sequential digestion would be better. Also more detailed information will be better trouble shooting

Generally I am suspicious if I get just one clone. You might better set up a new ligation reaction or do a transformation if you have got some ligation mix left

Ii mean to sy tat I got many transforments in tat i got only one right clone. It was done with my old ligation mix which was giving more background colonies. I digested the lig mix with sma1 and transformed for blue white screening. And i done PCR , i'm getting higher molecular band, .8 kb is my insert i'm getting around 1.3 kb fragment....?

One more experiment i follwed as u told to purify vector after CIP treatment , went for ligation with insert in 1:20 ratio and transformed. I kept CIP treated vector ligation and CIP untreated vector ligation and transformed as a control. But i coudnt get clones on VIL plates,.