Hi Prabuddha..

There seems a problem with the template amount when you are trying to reamplify from the purified template...try to do a dilution (1:10, 1:50, 1:100 etc.) of your gel purified template and do PCR again...you'll definitely get good product when you take less template DNA...all the best

Hi Prabuddha..

There seems a problem with the template amount when you are trying to reamplify from the purified template...try to do a dilution (1:10, 1:50, 1:100 etc.) of your gel purified template and do PCR again...you'll definitely get good product when you take less template DNA...all the best

Hi Ajay, thanks a lot for your reply. I have diluted 15 times in 2nd attempt. In first attempt I used 1 ul directly. But in all the nested PCR it gave big sized smear. Should I check with the dilution you have mentioned? But I have used less dilution earlier for short templates like 1.3 kb from cDNA etc, but did not get any such kind of problems. Is this because my current template is big sized and from genomic DNA? Please suggest more....

Hi Prabudhha

If your product in the first PCR was in good amount...you may have to dilute further.....this is one of the option that may help in your case...(as the smear right from the well is generally seen if the template is high).....but other possibilites may also be tried...like optimizing Tm and othet components of the enzyme....

Additionally...If your gel has been exposed on UV trans for a long time that can also danmage DNA, which can further affect in downstream PCR....so what you can do in such as case is load produc in two lanes....stain and view under UV one of them and isolate the band from the other corresponding lane....it may help

bye

normally, i used template 50ng / 50 ul pcr reaction. for the 1st, PCR if you got only band, it isnt neccessary cut the gel. just pure by using pcr (Quigen kit, pure from PCR). Pure by gel cutting, your pcr product will lose.

Your PCR size is 3.1... how many percent gel that used? 0.8 or 1 is ok.

Smear band.....your PCR got DNAse??? or not. Try to check,,, the specific and contamination with DNAse.

Cheer!!!

I can suggest that after purifying the band from gel once again perform gel electrophoresis to confirm the presence of template and take the amount of dna based on the intensity of the band appearence

Can you see a single band (a single PCR product) on your gel after the first amplification? If you have a single PCR product after the first amplification (by using the UTR primers) you surely do not need and do not want to purify it the amplification product from the gel. Just get a PCR purification kit or use the purification method with Ammonium acetate and ethanol.

@ Ajay, I agree with you with the serial dilution. Serial dilution are good if he wants good amplication for his gel. @ Claudia, I was wondering could he use Ammonium persulfate as well?

to Jefferey Freeman, Ammonium persulfate is not good.

1. Qiagen gel purification kit is notorious in introducing high salt and ethanol cross contaminations. Why do you need to gel purify your PCR products prior to reamplification. Can you just take your primary PCR reaction (take a microliter or so), dilute it 1:100 to 1:10000 and subject it to reamplification?

2. Some pfu types PCR polymerases are very difficult to work with. In the past I worked with Platinum pfx from Invitrogen. I would either get the product nicely or get nothing at all.

3. Since your reamplification fails, is it possible that the product is not what you expect it to be (i.e. your hypothesis is incorrect). This would explain failure using nested primers but the products should be reamplified using the same primers.

4. At this point I would try different polymerase, less demanding that the pfu. I understand you need fidelity, but it is also important to see if the products can re reamplified with any PCR polymerase..

here is the protocol for ammonium acetate DNA precipitation (it will remove primers' dimers and proteins, it is good for further amplification or Sequencing reactions)

1 VOL of your PCR of 4 M ammonium acetate (the final concentration should 2.5M)

2.5 VOL of your PCR of 95% Ethanol

at -20 C degree for 30 min

Centrifuge at max speed for 20 min

remove carefully the supernatant without touching the DNA pellet

Add 200 ul of cold 70% Ethanol centrifuge 15 min at max speed

Remove carefully the ethanol and let dry the pellet

re-suspend in water or 1X TE buffer

Hi, plz. Learn and discuss about:

1. annealing at lower temperature and increased time

2. addition of TritonX100,

3. Touch-down PCR , particularly for ur 2nd PCR

Hello every one

Dear Prashant

your first & third suggestions are illogical. It can't be reasonable for eliminating background.

Hi! First confirm after elution that your product is safe, just by loading little.

Then go for PCRs. Once your template is fine your PCR should work.

Thank you all for your helps. I am trying to use Taq DNA this time....2ndly, there were background smears along with the desired 3.1 kb band, so I had to go for the gel extraction procedure. And 3rd ly I have got all the nested PCR positive when I have just diluted PCR products and done downstream nested PCR. It went like this....1st round PCD from gDNA, I got smear, 2nd nested I got 3.1 Kb, 3rd nested I got desired band, 4th nested dgot desired band and in good amount.The amount of PCR product increased in each downstream PCR. The I decided to cut the 1st band tham means the 2nd PCR and purify by Qiagen kit. With that one I tried to do PCR with same primers and with the nested ones by doing 1:15 times diluted templates, I got big smears. Now I have used Taq instead of pfu to check if it can amplify the same. And tomorrow will get the result. And lastly decided to cut and purify the last nested product and clone....as that one seems to be more accurate as that is the 4th nested product.....So, please suggest if I could explain the problem...And I already know the annealing and other concentrations for which the primers already worked, so after purification why should I manipulate with all these parameters? Tomorrow I will also try to do PCR with 50 or 100 times diluted template.

I have checked with the template by doing some restriction digestion experiments. But in my opinion the DNA was very less eluted, so decided to do re-PCR. But in the RE experiments, I saw the quality of the cut products were not good. Thats why I asked whether Qiagen kit is capable of eluting big sized fragments without damaging it or not as I already have used the kit for purifying 1.3-1.4 kb fragments without any problem. But here the size is much bigger than those.

Hi! I am not aware of Qiagen kit, I used Geneclean kit it was perfect.

See let it be any kit,Go as per the instructions then it should work. First of all you are telling you are not happy with your product then how can you expect good results Prabudda. Just do it carefully every thing work fine. Good Luck.

@ Claudia, thanks for correcting me about the ammonium persulfate. You are suppose to use the Ammonium persulfate when performing a Western Blot.

Hi Prabuddah,

I don't know what you are planning to do with that fragment but be aware that the more PCR you do the more amplification mistakes you will risk to find in the seq of that product. If you are planning to clone it and to express it in some cell line you better check the sequence of the clone.

Then,

the extra bands that you see are bigger or smaller than the band you need?

A good trick for long PCR product is using a very short annealing time, like no more than 10 sec.

To eliminate higher bands a good trick is to reduce the elongation time (or extension time) to max 2 min for your product. Polymerase enzyme are way faster than one can expect.

I dont see any bands. only smear. and this is a genomic fragment of the gene sequence I got. I want to do intron analysis.

just to know.. how long is elongation step in your PCR?

As the specifications state that pfu synthesizes 1 kb per 2 minutes, I keep it 8 minutes elongation step in the PCR.

...it should be enough.. I'd try to diminish pfu concentration in the mix.

I use 1.25 units per reaction....

Maybe you want to think about changing your primers. how long are they? Be sure you can pick primers that work well at a temperature around 60-65 C degrees. 8 minutes for 3 kb is a lot of time. You will end up amplifying a lot of undesired products, especially bigger that yours. Most of enzyme are said to amplify 1kb per minute and that is already an overestimate.

Hi! I think your extension time is too much. Even to amplify 2.5Kb fragment it hardly needs 2 minutes. Go through the following links and modify your procedure. You may get better results.

http://www.fgsc.net/Aspergillus/Oakley_PCR_protocol.pdf

http://stanxterm.aecom.yu.edu/wiki/data/Product_manuals_attach/pfu.pdf

But fermentas recomends 1 kb per 2 minutes for pfu....

Try Takara Taq.

Your first answer was the best (Ajay Saini). By adding purified product to the PCR reaction, your template concentration is so high that you need only 5 to 10 cycles to produce a very large amount of the correct product. If you then continue the PCR for another 20 to 25 cycles, you will get "hyper-PCR" products which are illegitimate fusions of higher and variable molecular size. So either stop the PCR after a small number of cycles (try 2,4,6,8) or massively dilute the purified template-- 1/10,000 or even more--before adding it to the reaction! The reduced cycle approach may have the advantage of reducing the likelihood that any clone you get will have a PCR-induced mutation. Best might be to dilute about 1/100 to eliminate any salt or impurity carryover and then try 10, 12, 14 etc cycles of amplification.

Check that the Qiagen kit you use is suitable for the purification of the Size of PCR product you want to purify. Just in case : When you elute your PCR product from the Qiagen column, don't use water. It is also better to use TE instead of the buffer from the kit (If you want to reduce the risk of degradation of your template ). If you then dilute your template enough for PCR , EDTA from TE should not be a problem. After purification you can also check on gel to see if you have your band or a smear. You will know if it is a purification problem or the next step of PCR. I also think that 8 min as extension during cycling condition is too long for 3kb, try 4 + few seconds at each cycle. However, this is probably not the problem because it has work for you in the first place.

Hi Prabuddha, Few advices I would suggest:

1. Amplify from genomic DNA with pfu and then add A-overhangs with Taq polymerase.

2. TA clone this PCR product and through miniprep take good quality plasmid

3. Amplify your gene now from TA plasmid.

When you re-amplify gene from purified PCR product, It becomes difficult for pfu to re-amplify because of the salts etc. added during purifying and eluting process which hinder the polymerase activity.

ALL the BEST!.

Hi Prabuddha,

Did you run your purified product on a gel? Sometimes there is a problem with the columns and the PCR product is very low.

For me you have trouble with the template amount and you can do dilution of your gel purified template and try to do PCR again, the first comment for is right. All the best.

I have diluted the purified template upto 100 and 200 times and got a light band along with heavy background smears (The band is running a little slow here, may be due to the background smear). The band is better in 200 dilution than the 100 dilution. But when I diluted the template 400 and 600 times, and did PCR with pfu and deep vent, but only smear seen, no band.... And I have run the purified fragment in gel, it is perfectly alright. The band is very good. I cant figure out the problem. Its so confusing...

It might be the Qiagen kit causing the problem as Sarfraz sir has told....I am now going to clone it.

Have you tried reduced cycles?? Just set up duplicate reactions and when the machine has run for 10 to 15 cycles, hit pause and pull out one of the duplicates. If you see more of the good band and less of the smear, you can then try to find the right number of cycles that shows only the band.

I use 30 cycles.....Ok will do as you said...:)

How did you solve this problem Prabuddha? Can you tell me please