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PCR product purification


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#1 thegradstudent

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Posted 01 April 2004 - 05:38 AM

:rolleyes: 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
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#2 pcrman

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Posted 01 April 2004 - 09:57 AM

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

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 ul 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.

#3 kant0008

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Posted 01 April 2004 - 04:48 PM

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!

#4 thegradstudent

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Posted 01 April 2004 - 05:29 PM

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

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 ul 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.

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:
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#5 axisy

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Posted 27 April 2004 - 01:18 AM

EXOSAP ur PCR product

EXO1 nucleases & Shrimp Alkaline Phosphatase

#6 pcrman

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Posted 27 April 2004 - 06:36 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.

#7 carles

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Posted 28 June 2004 - 11:51 PM

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

#8 Ming

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Posted 29 June 2004 - 04:36 PM

Is the Wizard DNA clean-up kit well?
I'm trying! :(

#9 strial

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Posted 07 July 2004 - 12:26 PM

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.

#10 daniiiRNA

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Posted 12 July 2004 - 09:09 AM

There are still several problems possible: :o

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 B)

Once in our lab the load buffer was contaminated by nuclease. funny story...nothing works for weeks. :ph34r:

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

Best yield: Do PCR, aliquot 3-5Ál 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. :D

#11 BioAdventurer

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Posted 18 July 2004 - 03:01 AM

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

#12 mybioweb2

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Posted 23 July 2004 - 06:47 AM

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

Edited by mybioweb2, 23 July 2004 - 06:57 AM.


#13 ravibiot

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Posted 12 August 2004 - 09:55 PM

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

#14 postdoc2130

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Posted 19 August 2004 - 04:10 PM

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.

#15 aj_xy999

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Posted 19 August 2004 - 09:47 PM

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?




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