Dear Soundarya

how big are the insert and the restricted plasmids?

because sometimes those kit lode efficiency with big fragments? >8-10kbp

Manuele

The insert size is around 1.1kbp and the restricted vector size is 4.6kbp. The fragment is not so big though Manuele Martinelli

• Agree with Manuale about size restrictions
• Other than that yes you can: These kits are designed to extract DNA its just they are sold for a specific purpose
• But it should work fine for your restriction fragment
• A while back I used to clean them up with phenol and chloroform and also spin columns (for cloning) similar to what you are proposing

After melting or dissolving the gel slice on the chaotrope solution, try diluting it 1/2 before loading into the column. Also, what buffer system are you using for electrophoresis? If you are using Tris-borate or borax, try switching to Tris-acetate (borate is supposed to form complexes with cis-diols on the sugars of the agarose polymer).

Hope it helps!

Hi, I suggest the Alejandro Martin answer good for this question

I am using TAE buffer to run my gel Alejandro Martin

If you are cloning PCR fragments from non-phosphorylated primers I would suggest the following routine.

First ensure that your destination vector has cut by viewing a small portion on a gel. With the remainder of your digested destination vector, remove the 5' phosphates (using CIP, or Antarctic Phosphatase or similar), and perform a cleanup step (NOT a gel extraction) - I use nucleospin for this.

Then digest your PCR reactions with Dpn1 to remove the template (Dpn1 works nicely in Phusion polymerase buffer - but other polymerase buffers may not support it and require you to add some Cutsmart buffer before it works, so check). Next digest your PCR products with the appropriate restriction enzymes. Then perform a nucleospin clean up step afterwards (NOT a gel extraction).

Finally assess the molar concentrations of vector and fragment (nanodrop), and mix them in your desired ligation ratio (my preference is 4:1 insert:vector).

Perform your ligation / transformation as usual.

This protocol avoids gel extractions, which how ever you try them are always inefficient (ie always result in large loss of your DNA).

It works best if your destination vector has only a small drop-out between the two restriction sites (say 40 bp or fewer), as drop-outs this small are not retained by nucleospin (or similar) clean-up steps. Larger drop-outs may be retained, but should not clone as they will have been stripped of the 5' phosphates. Only the desired, double digested PCR fragment will have 5' phosphates in tact, and should be the only insert that can be ligated.

In most cases, the purification kits have a size range around which you expect to have a high concentration of eluted product. If you are working with high Molecular weight plasmid, I advise that you increase the time the product is on the column before the final elution. Alternatively, you could warm the elution buffer to about 65 degrees before the using it for elution.

Dear Soundarya

however, if the fragment that you remove from the plamid with the digestion is small (eg 20-150bp ) you can certainly perform directly PCR clean-up instead gel extraction because this kits are generally designed to remove small fragments as the oligo. If your fragment is bigger that 300bp than you need to perform gel extraction to separate it from the digested vector and minimize the risk of re-ligation of it into the vector.

Manuele

Manuele Martinelli My restricted fragment from plasmid is only ~70 bases. So i can clean my restriction product without running them on gel?

Yes! of course!!

it is convenient run a small amount of digestion on gel to check if it works. the only risk is that of you have some not digested pladmid, you will purify with the digested and during after the ligase you can find some empty colonies due to it, but if your digestion work well you can certanilly do it.

your pcr screening will show you of you have problem of backgronud or not and in case screen more colonies.

you can do it just performing pcr directly on your e.coli colonies ( if

you are interested yuo can check ProteoCool 4 at page 1 of my blog:https://proteocool.blogspot.com/)

i suggest to you to perform alkaline phosfatase digestion with cip just after digestion and than purify it with pcr purification kit.

good luck

Manuele

A dropout of 70 bp may be captured by a silica cleanup step, it is a borderline size according to the manuals. Nucleospin kits (see first link below) claim they can bind 50 bp and greater, while the qiagen kit linked next suggests fragments need to be 100 bp or greater to bind. So pick you kit carefully.

It should work for you though, as binding efficiency for a 70 bp fragment will be low, and of course you will have dephosphorylated it, so even if it is captured along with your cut vector it should not ligate back in.

When you do your ligations carryout out a control ligation with just the vector, and transform the same quantity of that as you use in your main ligations. That should show you what level background, if any, you have.

Good luck.

https://www.mn-net.com/Products/DNAandRNApurification/Cleanup/NucleoSpinGelandPCRCleanup/tabid/1452/language/en-US/Default.aspx

https://www.qiagen.com/gb/shop/sample-technologies/dna/dna-clean-up/qiaquick-pcr-purification-kit/#orderinginformation

The same kit can be used to purify PCR product and Gel Extraction.

But in case of Gel Extraction, you need to melt the cut gel band in the binding buffer (refer the kit protocol) and then follow the same procedure as that of PCR Purification.

If you are getting problems in the concentration of extracted plasmid concentration, try using 0.7 or 0.8 percent gel for gel extraction.

Take care that you are not adding hot binding buffer in which the gel has been melted onto the column, as it will reduce the binding of the plasmid to silica column.

Additionally, you can use hot elution buffer/water (60 degree C) for elution to further improve the yield.

John Hinks, for what it's worth, I once misplaced the right kit and in a hurry, had to purify a 65 bp dsDNA fragment using the Qiagen PCR purification kit you mention above (the one mentioning 100 bp as smallest recommended size). I don't remember exact figures now, but yield was quite acceptable, certainly more than 50%.

Cheers!

Thanks Alejandro, good to know! It's so often the way that we discover these things by accident. It sounds like Qiagen were hedging their bets in the manual.

John Hinks:

Hedging their bets, indeed. They sell a "nucleotide removal kit" -the one I misplaced- exactly for cleaning up less-than-100 bp DNA :-)

i dont know which specific thermo pcr clean up kit has Soundarya because

the maximun size of the fragment that can be removed change kit by kit.

Thermo for example suggest that its purelink pcr purification kit https://www.thermofisher.com/order/catalog/product/K310001

allow to remove fragments up to 300bp and from their datasheet (attached) seems to work at least up to 232 no

Manuele