PCR product purification-Molecular Biology

互联网2008-07-08

4211

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

-thegradstudent-

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I've used qiagen kits a lot and never encountered similiar situation.

QUOTE

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

Did you run a gel before purification to make sure your PCR amplification was successful? I usually have two type of kits at hand, one is the PCR purification kit, the other is Gel purification kit. After each PCR, I run 4 μl of product to see what my amplification looks like. If there are strong primer dimers or non specific bands, I will run the whole PCR reaction in another gel and cut the bands followed by purification using the Gel kit; if the amplification is clean (desired bands are strong, no primer dimers or non-specific bands), I will go for PCR purification kit directly w/o further gel purification.

Hope it helps.

-pcrman-

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Hi!

I have never tried Zymogen kits, but Qiaquick works well normally. A few possible problem areas:

1) How big is your PCR product? Has to fall within the limits specified in the kit

2) Qiaquick is the gel kit, right? So you actually have to cut out your product. How intense was your band of interest? Maybe your PCR wasn't particularly good

3) Check the pH of your elution buffer, or (especially!) water, DNA will not be eluted if pH is too low, has to be slightly basic. (MQ water is not nevessarily neutral)

4) How old is your isopropanol? It's hydrophilic, so if it gets opened too often, it will be "diluted", so your concentrations will change. Get a new batch

Hope this helps!

-kant0008-

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QUOTE(pcrman @ Apr 1 2004, 09:57 AM)

I've used qiagen kits a lot and never encountered similiar situation.

QUOTE

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

Hope it helps.

Thanks for your notes. My PCR product is 525bp. After every PCR I always run a gel to see if it was successful. In some cases, not always, I have intense bands, along with smear and primer dimers. Really, the QIAquick kit I had used is old, so I will take the advice of getting a new batch.

Thanks a lot guys!!

Thegradstudent:rolleyes:

-thegradstudent-

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EXOSAP ur PCR product

EXO1 nucleases & Shrimp Alkaline Phosphatase

-axisy-

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QUOTE(axisy @ Apr 27 2004, 01:18 AM)

EXOSAP ur PCR product

EXO1 nucleases & Shrimp Alkaline Phosphatase

I like ExoSap too. I knew it because one of my colleague used it. It's a great product and can save you lot of time. But your amplification should be clean to use it, which means there is no nonspecific bands as revealed by a gel run.

-pcrman-

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i used ultraclean gelspin DNA purification kit (Mobio) . It's very quickly, and efficient.

-carles-

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Is the Wizard DNA clean-up kit well?

I'm trying!

-Ming-

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Run your PCR product on agarose gel 1% check you DNA band.

If your DNA band is too low, pool 2 or 3 PCR and run on agarose gel for purification.

Use the minimum agarose% to minimise lost of sample.

I'm using Qiagen minielute and it give me great results in a range of 400pb to 3 kb.

-strial-

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There are still several problems possible:

your solution is to old because it is diluted (already said)

500bp is kind of small. Use 2% agar. There are several special agar- powders for high concentration. You do not need them. Don't waste your money. Use the normal on...it works fine

Once in our lab the load buffer was contaminated by nuclease. funny story...nothing works for weeks.

Other story. Once they exchange the UV-lamp. Somebody turned the power to high...the bands get damaged by the lamp and disappear

Best yield: Do PCR, aliquot 3-5� and check by gel, if PCR was successful go on for purification by Qiaquick. Best yield...no gel cutting, no loading buffer and no UV lamp. Takes 10 min. Actually I love it.

-daniiiRNA-

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In my point of view this doesn't look like a problem with the kits you are using, but something to do with the gel conditions. Please, double and triple check that your buffers and gel conditions are at least sufficient to obtain a clean sample. GEL ARE TRICKY!!!!! A very good alternative is to ask a collegue to give you some of his/her sample (positive control, or you should have some allready) and check out how this is shown in the gel, and what is the purification yield, if any!

Try this if it seems logical to you, and tell me the aftermath

See ya!

Yannis

-BioAdventurer-

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

-mybioweb2-

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hello gentle man,

i used to use promega whizard pDNA purification kit.

i hav used it to purify 256bp DNA product.. so no soubt to follow it.

u have to give more incubation time with the purification resin and hav to getly mix it for more than 5 min.

all the best

best regards

sincerely,

ravi

INDIA

-ravibiot-

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Qiagen's spin column formats sometime have this problem that bother you so much and you just don't know what's going wrong. Try Epoch Biolabs' GenCatch PCR clean-up kit (epochbiolabs.com), it's a better kit than Qiagen's in both consistency and price in our lab.

-postdoc2130-

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usally we purify directly from excised gel fragment using spin columns. this is followed by EtOH precipitation & re-dissolution.

that has always worked wonderfully without giving any problems. ever tried that?

-aj_xy999-

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Hi Guys,

I use Jetsorb from Genomed. It works every time with small and bigger fragments.

Good Luck

-haui-

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for PCR product purification used for cloning, phenol/chloroform's classical method is ok. I think kits are unnecessary.

-iamfat-

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I use the Qiaquick PCR purification kit all the time without having any troubles. Maybe your PCR did not work. Otherwise you can use a PCR puirification kit from Roche.

-Sphingoman-

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First look on agarose gel (2%) that you have PCR product of interest.

If you have low concentration you can pool some samples together (same kind of PCR product of course )

I normally use the PCR purification kit (Jet quick) from genomed Incorporation. I thinkl that one is working just fine.

I have purified PCR product that is 150bp.

I hope that you soon will purify youre sample succesfully.

/Nina

-Nina-

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Don't forget that many of the kits elute the purified PCR product in water or buffer. This generally dilutes your product, especially for gel bands! 100 ng of DNA will give a nice, bright band if the initial well in the gel wasn't very wide. Bind that 100ng to a membrane and elute into 50 μl of buffer, and you've got a concentration of only 2 ng/ul! Load 5 μl on a gel to check for product/quantify, and you probably won't see a thing. Eluting in less than 50 μl increasaes your concentration, but you lose some product in the process.

So, if you don't see product after cleaning, and think you've lost your product, my suggestion would be to ethanol precipitate, preferably with a co-precipitant such as PelletPaint (Novagen). Then you can see if you have a product, cause if you don't you also won't have a pellet, and you can get your concentration back up to where you can see it on a gel or quantify it by spec.

Just my 2-cents worth.

-Turtle

-turtle-

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I ususally purify three band, from the agarose gel containing the same PCR product, together to minimize the risk for diluting the sample.

/Nina

-Nina-

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seeing that your clock was ticking since april, not sure how much help my reply will be..

anywayz,

been using sod acetate- isoprop precipiation with no problems all the while.

good enough for cloning and gives good seq result when used as template..

good luck!

let me know if u need the procedure

-julianne-

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QUOTE

for PCR product purification used for cloning, phenol/chloroform's classical method is ok. I think kits are unnecessary.

For cloning of PCR products, does phenol/chloroform extraction necessary? or can I just do a precipitation?

My two cents: With using a kit, to increase concentration while get good recovery, I usually use 25-30 μl buffer to elute DNA, after elution, use the same elutant to elute the same column once more.

-postdoc-

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Thanks all you guys for your numerous replies!!!

I have just repeated the PCR and obtained a better band... Next time I have a pool of choices here!!! But I am absolutely sure that this helped a lot of people. That makes this site great!!

-thegradstudent-

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Sounds like good old fashioned ethanol precipitation would do the trick.

Sometimes the the oldies are the best!

-tuckern-

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This is a silly question, but I was just thinking today about how I use the Qiagen PCR purification kit all the time, but have no idea how it works! What do the buffers PB and PE do? I guess PB helps DNA bind to the spin column, but how? And why do you need to add ethanol to buffer PE? What does it do? Thanks so much.

-BuiltToSpill-

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Hi,

A colleague has just recently tried exozap and compared it to qiagen columns. Qiagen won big time!

Is there a trick to using this exozap? I hadn't really heard about it until this year... It'd be great if it worked as I have a very high number of samples to churn through and qiagen just gets expensive...although it's always worked for me, really well in the past.

Any suggestions/protocols would be appreciated.

Cheers

Maria

-MJC-

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QUOTE(Nina @ Sep 16 2004, 02:58 AM)

I ususally purify three band, from the agarose gel containing the same PCR product, together to minimize the risk for diluting the sample.

/Nina

[snapback]7169[/snapback]

Hi,

I was wondering if you've tried cleaning up with sod-acetate and isopropanol in a 96-well plate or whether the isopropanol makes the pellet too slippery for this to be done...a protocol would be appreciated.

Regards

Maria

-MJC-

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invitrogen chargeswitch PCR clean up

-John Buckels-

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Try Roche;s High Pure PCR Product purification kit. I have used it successfully for many years now, it has a few more steps than some of the others, but can clean up PCR's of sizes from 80bp to 1200bp.. (as far as i know). Good Luck!

-janbrisbane-

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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

-sarpetit-

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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

-Seda-

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QUOTE(aj_xy999 @ Aug 19 2004, 10:47 PM) [snapback]6679[/snapback]

usally we purify directly from excised gel fragment using spin columns. this is followed by EtOH precipitation & re-dissolution.

that has always worked wonderfully without giving any problems. ever tried that?

hi,

i usually purify PCR products directly using phenol chloroform extraction method,never faced problem unless the PCR product was too less in quantity and too small in size.

its easy and u can try for ur 500bp fragment.

Quaigen colum works fine, 've used that also.

if u need to know the direct elution protocol ,plzz do let me know.

thanx

-arin-

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QUOTE(mybioweb2 @ Jul 23 2004, 04:47 PM) [snapback]6197[/snapback]

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

Hi there.

I� trying to precipitate a PCR containing a 80 bp DNA fragment. I see the beands on the gel but after the precipitation i lose the DNA. Does anybody knows a protocol, not a kit, i may use to keep the DNA after the purification.

Thanks a lot

laurejosiane

-laurejosiane-

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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.

-jlee-

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QUOTE(aj_xy999 @ Aug 20 2004, 07:47 AM) [snapback]6679[/snapback]

usally we purify directly from excised gel fragment using spin columns. this is followed by EtOH precipitation & re-dissolution.

that has always worked wonderfully without giving any problems. ever tried that?

How long are you centrifuging in those columns? Gel purification kit columns cannot go over 3 min., otherwise your DNA gets stuch in the agarose mesh, so don't spin too long!!! Let the DNA air-dry really long after the EtOH precipitation, try to avoid 95 celsius heating if possible. Good luck!

-smoochiepie79-

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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.

-sChlOsh-

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QUOTE(jlee @ Nov 16 2005, 07:53 PM) [snapback]31499[/snapback]

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

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.

-Pitono-

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QUOTE(thegradstudent @ Apr 1 2004, 03:38 PM) [snapback]5253[/snapback]

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

Sureclean from Bioline is a superb product. Highly recomend it for this function

-u03cka-

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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.

-Pablete-

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QUOTE(Pablete @ May 19 2006, 06:15 PM) [snapback]52462[/snapback]

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.

How did you try to 'add an A'? That's usually the most difficult step.

James

-jamesp-

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I like very much the clean up kit from Macherey-nagel. It works fine for me!

-Raffaela-

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i am using RBC pcr gel purification kit. After purification when i check to see my band there is hardly anything visible despite beautiful amplification. suggestions please.

-garimamahlawat-

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