Hi Rachel, about your first question you need to gel purify your digestion to control the digest reactions, eliminate the digestion enzyme and buffers and to remove undigest plasmid.

You said that you digest your plasmid only with BamH1, did you perform a dephosporylation? In a single digestion reaction it is really easy to have self-ligation. Also in this case you have to perform a gel purify to remove the dephosporylation enzyme and buffer.

For the second question, yes! You can negatively affect your ligation, in your reaction you have the digestion enzyme and buffers plus your ligation enzyme and buffers...

In my experience I always gel purify fragment and plasmid. I din't have problems with agarose residue, follow the instruction of gel purify kit.

In my humble opinion follow the rules for the correct cloning and you will prevent wasting your time and your material.

Good luck

Hi Valeria and SM, thanks for your responses.

Valeria, no I did not perform a dephosporylation. Is this a necessary step?

I was told to use two different buffers for my digests on my amplicons and plasmids and never told to do gel purification or dephosporylation on either of them. 

So I will need to first dephosporylate my plasmid and then perform gel purification on both my amplicons and my plasmid, correct?

Again, thank you for your help. I appreciate it.

Hi Rachael,

In my experience, it is always best to gel purify any fragment/plasmid you wish to clone because buffers can definitely have an affect on cloning efficiency.  It's a little upstream insurance for downstream applications and you can immediately rule out buffers etc from any cloning "issues" that you encounter later on.  

Dephosphorylating your vector is recommended to prevent self-ligation and ensure a higher cloning efficiency.  Again, its a quick bit of insurance to increase your chances of ligating your insert, and becomes an absolute must if you can't do blue/white cloning.  

To answer your question, cut, gel purify, and then dephos your vector.  Cut, and gel purify your insert(s). Ligate and transform.  Good luck! 

Hi Barry,

Thanks for your answer! That sounds like a good idea. I have talked with my advisor and he said that since I have such a low amount of DNA that I shouldn't gel purify my fragments because I may lose some of my DNA in the process. But I do plan on gel purifying and dephosphorylating my plasmid. Thanks again! :)

Hi Rachael,

If you have a low amount of product from your PCR, you can always perform multiple pcr reactions and then pool the products (using an error correcting enzyme of course to minimize amplification errors) prior to RE digestion and gel purification.  You only need a few nanograms of vector and insert so even if you loose 10-15% in the gel purification, it typically doesn't pose any problems with cloning.  Good luck!

At least cofirm the ligated produc be redigestion by bamhi individaly, bgli individual and both together. those 3r reactions would results in 2 linear form and the compination should result in the release of your product and then you can propagate these colonies again as sometimes there are false poistives. and yes gell extraction is important. youbcan also

Hi Rachel, Barry just suggested you the best thing to do, the dephosporylation is really important if you digest with only one enzyme, the self-ligation is assured!!! If you will use, in future, two enzymes you can avoid it,  you have a reduced self ligation, I often didn't perfom dephosporylation in this case. For the amplicon it is not necessary because, in general, they are shorter than the plasmid so it is difficult a self ligation.

Your advisor points out a problem but as Barry said you can prevent the amplicon low amount with more PCR, and for your plasmid, you have to amplify again with a midi or maxi prep so you have a stock of your plasmid. 

When you perform a cloning reaction:1) you need a good amount of materials, 2) you have to digest a good amount of DNA without exceeding, or increase the time of digestion (for example ON, I talked about the plasmid), 3) perform dephosporylation when neeeded, 4) gel purify again if you perform dephosporylation.

Gel purify seemed only a waste of time and material but if you don't perform this you have the undigested plasmid problem (one of the first cause of failure cloning and screening of false positive mini prep) and to eliminate the salt buffers of restriction and dephosporylation reactions. They affect the ligation enzyme activity that need its own buffer so its own salt concentration.

Hope the best cloning to you :)

Hi Barry, yes, I ran multiple PCRs and had to pool some of my products together (to get the proper amount for digestion). If I only need a few nanograms, then I think I will be ok with gel purifying my amplicons.

Hi Valeria, so you suggest gel purifying after the dephos? That makes sense so you can get rid of the buffers. 

Thanks again everybody :)

Hi Rachel, some dephos enzyme can be combine with digestion or immediately after, depend on the kit and the vendor, to save time. Yes I recommend you to gel purify after dephos.

Awesome, thanks again for your help!

Hi Rachael,

I agree with Valeria.

Generally To purify the DNA fragments after the digestion of your plasmidic vector is a good way to proceed. You can select the right size for the fragments needed for ligation.

About amplicons' purification, the problem that you may face if you don't make a gel purification is the following one: your template vector will still be in your mix, even if your buffers are compatible with your REs and ligase.

You will have a lot of unrestricted vectors in the mixes, competing with your ligated plasmid for a transformation.

So, when screening for transformed bacteria (for example), many (even most) will be transformed with the original unrestricted vector.

That is the reason why a gel purification of both the amplicons and the correct fragments from your vector (generally the one with the antibiotic resistance) is required.

I hope this answered your question.

Hi,

Instead of gel purification, can PCR purification be done for RE digested Product?

yes

dear researchers,

I have a 16 kb linear plasmid which looks stuck in the column, and I can not neither cut it from gel nor even clean it up directly. is there any solution for eluting this fragment from column or can I perform ligation directly without cleaning Up the digested vector?

Dear Ashkan Alamdary ,

The magnetic bead based purification can serve your purpose.

Here is the link below for BD for the magnetic beads.

https://www.beckman.de/reagents/genomic/cleanup-and-size-selection/pcr/a63881

Its not necessary that you only chose BD there are other companies also

You can follow the question below to have some useful information for your protocol.

https://www.researchgate.net/post/Can_I_use_Agencourt_Ampure_XP_for_genomic_DNA_purification

Good luck!

Hi,

Dr. Barry's response is accurate. It is recommended to perform a gel purification so as to avoid the buffers and other contaminating impurities from hindering the ligation process.

Yes Rachael you need to gel purify plasmid

Can someone let me know how long can i store the purified double digested plasmid/DNA? Is it okay to use after 3 months for ligation?