Hi, you can try:

1. To increase T° annealing (as suggest by Pero) to have a more selective amplification.

2. To redisign your primer according your preference in order to obtain selective annealing and to avoid nonspecific fragment (eg. more long primer , 50%GC, G or C at 3' end..etc..)

Are your multiple bands near about them in terms of bp? If yes, it's possible that are the same fragment but with a little of difference in bp due to polymerase slippage.

I hope to help you, good luck

The bands are all over the place. Lots of them too. All the way from 5.5kb down to 50 bp. I tried a gradient of annealing temps for 50 to 70 and nothing helps.

please try first to make hot start denaturation of DNA Before amplification and chilled on ice

put an amount of DNTPs equivalent to your product size

USE an post extension time to remove any of UN extended product

ALso taq polymerase 3unit in a reaction tube ....other meaning try diffrent concentration of taq polymerase

AT rich regions can be difficult, but are not impossible. Just from the information you posted, I'd say there are at least two issues.

First, Pero is correct. You will likely need to try several different annealing temperatures. Low temperatures are a problem for several reasons. First, mis-priming occurs more readily at low temperatures. Second, low annealing temperatures can promote formation of secondary structure. All of this means that you end up with a lot of fragments instead of the single product you are looking for. So if you haven't tried a higher annealing temperature, I would definitely give doing so some thought.

The general school of thought on annealing temperature is that you want a temp that is about 3 to 5 degrees lower than the Tm of your primer. For AT rich sequences some folks look for areas of long stretches of A/T in their desired product and try to design an annealing temp based on the supposed melting temp of that region. The problem with tailoring your annealing temp to a small region of your overall desired sequence is that you end up ignoring some basic rules of thumb for producing successful PCR reactions. Additionally, the behavior you get from a section of A/T rich DNA as a part of a longer sequence during elongation is much different than what you would get from an A/T rich primer so the comparison doesn't hold up.

Second, you will also have to find an optimal elongation temperature as well. 60 degrees seems a bit low. Remember that Taq activity is very temperature dependent. At 60 degrees the activity of Taq is about half of the activity at 70 degrees. So you want the elongation temp to be as high as possible, i.e. as close to 68-70 degrees as possible. This will increase the efficiency of your PCR reaction and result in fewer fragments as well. Alternatively, you could go for a longer elongation time, but at 5,000 bp I am betting that you will also need a higher temp to get the job done.

If you insist on going with the lower temps, then I would also re-check my primer for secondary structure formation.

If it were my experiment, I would find a master mix I like (I prefer OneTaq 2X Master Mix from NEB, cat # M0482S) and I would start at a standard 94, 55-58, 68, run the product on a gel and see what I got, then adjust as needed. It may be that you get a decent yield of your desired product, with some other fragments/bands, and you can just cut out the band you want and call it a day.

Anyway, I hope this helps.

Good Luck and have a great day!

What is your current PCR profile? What are your primers?

CM

Hey all,

1. You need make sure your template is good( set up a positive control).

2. Need make sure your target sequence is correct,also check your primers.

3. Primer design( lots ppl give very good advice). I would say your need design at least 2 primers from each side. Then go 2 round PCR, first using outside primes at lower PCR condition, 2nd round use inside primers( you also can try difference annealing temperature). Most time first round you may get multiple bands, but after 2nd round just 1 band left or strong right size band.

4. Try difference polymerase( from difference Co.). You can see the big difference(I did this before).

5. PCR machine. Can set up muti- annealing temperature at same run.Annealing temperature can start from 50,52....... 60, ext. follow difference polymerase.

Hope this helps.

Did you try any of all answer....please back and give us feed back...thanks

Good point Pero. That final elongation step is critical to successful PCR. If you are running the elongation step at 60 degrees you will need a final elongation step at least twice as long as the typical rule of thumb.

Yes, please provide feedback.

You need change your PCR profile That is denaturation is for 1 min

annealing temp is also for 1 min

and the elongation is for 2 min

because of your product is big (5 Kb). so every step need bit more time.

so please try this and give feedback.

I am on vacation right now and cannot work on my bench. I thank all of you for your helpful responses. I found a useful paper on amplifying AT rich regions. Betaine was used. Choice of polymerase is important. Please explain to me why the final elongation step is so necessary please!

Final elongation step is important especially in long product size if you have any product with short ended not complete its full length this final long elongation step will perform this step but you must select a taq polymerase enzyme fully active like gold taq and with a concentration of unit equivalent to this product size

I will try first annealing the temperature at 60, 62, 64, until 70 and I would make sure that the final elongation is minimum 30 minutes.

Many factors to consider here. Is this a repetitive DNA region? If the primers are annealing to repetitive sequences, then no matter what, you are going to get multiple bands. If that is the case, you may want to go slightly farther away and design unique primers. Alternatively, if you are getting the right amplification product, which should be the highest band, you can gel purify that for downstream applications.

Another important consideration may be to increase the preponderance of one of the target strands by performing an asymmetric PCR. Use only one of the primers for 15-20 cycles resulting in linear amplification of one of the target templates. You can then add the second primer to hopefully improve the amplification efficiency. I would do two parallel reactions using only one of the primers in each for initial linear amplification and then continue regular PCR by adding the other primer. This way you are taking care of the possible mis-priming of one the primers during the initial cycles.

Hi dear Gary

You must run temperature and MgCl2 concentartion gradient to find the best annealing temperature and MgCl2 concentration.

It consume more time than conventional PCR.

Cheers