PCR product purification - (Apr/01/2004 )

hey,

I always use Eppendorf's Perfectgel clean up kit. It s really easy and fast, and has always worked great for me...

good luck

Hi,
If you are trouble with kits, you can use chloroform extraction, this is perfect. Sometimes, colums are contaminated. Although, Qiagen is perfect, this kit also has some problems. At this point I advise chloroform extraction.This is so safely
Good luck

if you have your band at the expected size without other nonspecific bands, try EtOH precipitation if you are kits-phobia !!

I can't believe you guys wasting your time discussing about this. PCR purification, DNA fragment recovery from agarose gel etc have been the topic for a couple of in deepth reviews almost 10 years ago and the method can't be any easier. All those companies you people mentioned here using the same thing!!!! some of you may well work those companies and promoting your product, who knows!!!

Here is a very cheap and reliable procedure we have been using for quite a while and something you can trust and save a good chunk of money for more expensive equipments

Binding buffer:

5.5 M GuSCN (guanidine thiocyanate), adjust pH with a few drop of Acetic acid, or if you prefer, use 20 mM Tris buffer pH 6.0
This buffer is good for both PCR clean-up, Agarose gel solubilization, reaction buffer exchange, removing un-incorporated dNTPs in a labling reaction ect. The only issue is GuSCN is pretty expensive. We only us it for DNA fragment recovery from agarose gel. for other applications we use cheaper chaotropic salts.

Wash buffer:
75% EtOH. Or if you prefer 25 mM NaCl, 5 mM Tris-Hcl, pH 7.5

Silica Membrane Mini Spin Columns:
You can make these columns by yourself (refer to Ray Wu's 1987's review) Or you can buy these columns at large quantities for a good price from epoch biolabs.
We bought 5000 pcs spin columns from them and used for almost everything: plasmid prep, pcr cleanup, gel extraction of DNA fragment from agarose gel, total DNA/RNA, genomic DNA, buffer exchange etc.

Just my 2 cents for your reference.

Have problem with my gel purification and need help.

i did gel extraction and undergo purification using QIAquick gel extraction kit. I've got my DNA fragment (1.3kb). However, i also got some unspecific band (few smaller size than 1.3kb and two larger size than 1.3kb..!). So, i heat up the purification product to 90 Celcius. Now, i've got 2 bands: one is my DNA fragment and the other one is the unspecific band which is larger than 1.3kb.

May i know if anyone knows what happen to my purification product(since i've done gel extraction and logically i should get only 1.3kb band..)? And how can i eliminate that extra band ?

Thanks in advance.

I can't believe you guys wasting your time discussing about this. PCR purification, DNA fragment recovery from agarose gel etc have been the topic for a couple of in deepth reviews almost 10 years ago and the method can't be any easier. All those companies you people mentioned here using the same thing!!!! some of you may well work those companies and promoting your product, who knows!!!

Here is a very cheap and reliable procedure we have been using for quite a while and something you can trust and save a good chunk of money for more expensive equipments

Binding buffer:

5.5 M GuSCN (guanidine thiocyanate), adjust pH with a few drop of Acetic acid, or if you prefer, use 20 mM Tris buffer pH 6.0
This buffer is good for both PCR clean-up, Agarose gel solubilization, reaction buffer exchange, removing un-incorporated dNTPs in a labling reaction ect. The only issue is GuSCN is pretty expensive. We only us it for DNA fragment recovery from agarose gel. for other applications we use cheaper chaotropic salts.

Wash buffer:
75% EtOH. Or if you prefer 25 mM NaCl, 5 mM Tris-Hcl, pH 7.5

Silica Membrane Mini Spin Columns:
You can make these columns by yourself (refer to Ray Wu's 1987's review) Or you can buy these columns at large quantities for a good price from epoch biolabs.
We bought 5000 pcs spin columns from them and used for almost everything: plasmid prep, pcr cleanup, gel extraction of DNA fragment from agarose gel, total DNA/RNA, genomic DNA, buffer exchange etc.

Just my 2 cents for your reference.

I have tried to purify my PCR products by using 3 different approaches:

1) QIAQuick PCR purification kit: but when I run the gel to see its efficacy, there was simply nothing!! it cleaned everything including my band!!

2)Zymogen's Cleaning & Purification kit: Intact sample, concentrated but still dirty

3)Zymogen's Purification kit from Gel: It cleaned but the yield was too poor and thus unfeasible for further cloning.

Any suggestions!!! Any modification of these kits or any "labmade" recipe??

I will really appreciate your comments, the clock is running!!
Thanks in advance,
Thegradstudent

Hi guys. First of all, thanks everybody for you advices.

We are having problems in cloning a qRT-PCR product. We`re permorming a Taqman Assay and trying to clone the amplicon, just to sequence it; as you can imagine, we do not know the specific zone of amplification (pre-made assays advantages ) and it becomes neccesary to clone the fragmente just to use a general oligonucleotide to sequence and see the amplicon. Applied B. specify almost nothing.
We do know the size of the amplicons, and they are really small, they are rounding the 80pb. I am searching for columss capable to purify very well small products and with high efficency, because my product is small, but also in low quantity.

In first and "trytoseewhathappen" approach, we tryed to precipitate amplicons with Etoh, add an A to make cohesive extrems, and try to clon them in PgemT vector. It was a complete disaster.

Hope you understand the situation.
Any advice??

Regards form Spain.