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PCR product purification - (Apr/01/2004 )

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wacko.gif 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-

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 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.

-pcrman-

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-

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!!


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:

-thegradstudent-

EXOSAP ur PCR product

EXO1 nucleases & Shrimp Alkaline Phosphatase

-axisy-

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-

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

-carles-

Is the Wizard DNA clean-up kit well?
I'm trying! sad.gif

-Ming-

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-

There are still several problems possible: ohmy.gif

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 cool.gif

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

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

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. biggrin.gif

-daniiiRNA-

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