Hi Ray, as you know Taq polymerase is most of the time inefficient at amplifying promoter regions which are GC rich. I don’t really like DMSO. It doesn’t always work. Provided that your oligos are good, I would add betaine at 1.5 to 2.5 M final concentration. Nucleic Acids Res 25 (19): 3957–8 (1997). It has always worked! You can purchase it from Sigma. I would also use a Polymerase which has strand displacement capacity. Write back if its works!

Hi Ray, as you know Taq polymerase is most of the time inefficient at amplifying promoter regions which are GC rich. I don’t really like DMSO. It doesn’t always work. Provided that your oligos are good, I would add betaine at 1.5 to 2.5 M final concentration. Nucleic Acids Res 25 (19): 3957–8 (1997). It has always worked! You can purchase it from Sigma. I would also use a Polymerase which has strand displacement capacity. Write back if its works!

Thanks, Abdelakim. I think I have betaine on hand, so I'll give that a shot right after the holiday. I'll let you know how it turns out. Out of curiosity, do you know what betaine does differently than DMSO in the PCR?

Hello ray....!! You can use the red genomic taq of sigma that may be useful for you to amplify next 7 target promoter. As well as you can use betain solution to increase the specificity and efficiency of amplicons. Generally red genomic taq (sigma) is used to amplify very difficult targets. Hope this will work for you.......all the best.......

Hello ray!

You can use the iProof High Fidelity DNA polimerase (Bio Rad cat. 172-5301) if your target is GC rich use the GC Master Mix (iProof High Fidelity Master Mix cat. 172-5320) and remember always use DMSO (1.5 to 3%).

Hope this will work for you.......all the best

Hi Forest,

If you want to try something a bit different, you could give Recombinase Polymerase Amplification (RPA) a go. It's like PCR, but without the thermal cycling, so it goes really quickly (

I had good experience with Phusion DNA polymerase (NEB), using the provided GC buffer. When amplifying genomic DNA I usually zap the template with a low power sonication for 10 seconds, not enough to shear the DNA but enough to introduce a few nicks that help release tension allowing the polymerase to more easily displace the complementary strand.

Hi

I would start to wonder about the primers you have designed. You haven't mentioned how confident you are about the sequence information you used . Would it be the sequence of a single genotype of a model species? Are you carrying out the PCR using template DNA from the same genotype? Is there a possibility of sequence variation that is confounding your attempts?

good luck

My suggestion is in favor of Y. Lubelsky's post. I have obtained great results using Phusion DNA polymerase, and its high fidelity and fast processivity will allow more trials to be conducted in minimal time. I also recommend fragmentation of the genomic template. Unfortunately, the propensity for truncated products may increase. This is remedied with gel extraction of full length product after amplification.

Hi.

I had good experience with Amplification kit ,in which all the reagent come together.,means taqpoly,PCR reaction buffer,AmpliTaq gold,allelic ladder& control DNA.

Gud Luck

Hello, I am more specialized in primer design than in wet biology.

First thing, BLAST is not a good software for checking primer specificity. Generally, Bioinformatician uses clustering software like usearch or primer_validator to deal with that.

Second thing, did you ckeck the cross-linking/autodimer formation for your primers ?

Last thing, how did you compute the theorical Tm ?

Best,

Julien

In order to minimize the error component u can try this........

High-fidelity DNA synthesis conditions are those that exploit the inherent ability of polymerases to discriminate against errors. This review has described several experimental approaches for controlling the fidelity of enzymatic DNA amplification. One of the most important parameters to consider is the choice of which polymerase to use in PCR. As demonstrated by the data in Tables 2 and 3, high-fidelity DNA amplification will be best achieved by using a polymerase with an active 3'-->5' proofreading exonuclease activity . For those enzymes that are proofreading-deficient, the in vitro reaction conditions can significantly influence the polymerase error rates. To maximize fidelity at the dNTP insertion step (Fig. 1A,B), any type of deoxynucleoside triphosphate pool imbalance should be avoided. Similarly, stabilization of errors by polymerase extension from mispaired or misaligned primer-termini can be minimized by reactions using short synthesis times, low dNTP concentrations, and low enzyme concentrations. Additional improvements in fidelity can be made by further manipulating the reaction conditions. To perform high-fidelity PCR with Taq polymerase, reactions should contain a low MgCl2 concentration, not in large excess over the total concentration of dNTP substrates, and be buffered to approximately pH 6 (70 degrees C) using Bis-Tris Propane or PIPES (Table 2). These buffers have a pKa between pH 6 and pH 7 and a small temperature coefficient (delta pKa/degree C), allowing the pH to be maintained stably throughout the PCR cycle. For amplifications in which fidelity is the critical issue, one should avoid the concept that conditions generating more DNA product are the better conditions.

Can we use Pfq DNA polymerase instead of Taq??????

Hi,

before trying different polymerases I agree with some other that you should check some basics:

1) Have your primers strong secondary structures o primer dimers that prevent your PCR reaction?

2) Are you sure about misparing bases due to genetic polymorphisms?

Once you both questions checked you can try to optimize your PCR to be more "permissive". That should work if you actually don't have inespecific interactions as your blast indicates.

1) Decrease your Tm (to 55ºC or so)

2) Increase your primer concentration

3) Increase your MgCl2 concentration (you could try 2mM, 2.5mM or even 3mM)

4) Try nested PCR and touch down strategies

Those are cheap, easy and fast tries.

I hope it helps!

Hi Forest,

All the responses are worth trying, but problem is they are lot and will take a lot of your time. I will suggest trying to lower down the Tm or do a gradient PCR for your rest of the amplicons you are not able to amplify. Your Tm may be too strict with rest of the amplicons. So I will suggest trying a gradient PCR with Phusion DNA polymerase with high GC buffer. And wish you good luck!!!

Hi Forest,

In my understanding, the first time you need to check the specificity/dimers/hairpin of your primers whose not worked. The Tm difference of 3 degrees between forward and reverse its not so good. After that, as you already altered the mix and are using a excellent DNA polymerases, you can try change the cycling times, like denaturation, annealing and extension times.

I hope helped you.

Best,

Washington

There is a simple but efficient program to see if you can have dimers with your primers, it's the auto dimmer. You can get it on the following link: http://autodimerv1.software.informer.com/

Hi Forest,

I would give my vote to the betaine + a high processivity polymerase. Promoters are quite CG-rich and an enhancer solution sometimes makes the difference. We usually prepare an enhancer solution 5X (dark 4ºC) with betaine (Sigma B-0300) 2.7 M + DMSO (Sigma D-8418) 6.7% + DTT 6.7 mM. You can try 2x, 1x, 0.5x concentrations in your reactions to see which one work with each primer pair. Hope this helps!

Hi Forest..

People have given lots of suggestion but u cant try all ....I have amplified till 6 kb of length with Qiagen kit. Plesae one try it and optimize it with gradient PCR. one thing more plz check your primer once again by amplify software...there should not be any nonspecific binding.

Hi Forest!

I am 100% on the opinion of Henry N. You have to check your primers and the template DNA. Do not waste your time with different polymerases.

Hello,

Try with decreasing your annelingt température!

In mix, you CAN add BSA instead of dMSO solution

or nested PCR

Good luck and good work

Hi Forest

I vote up for the gradient PCR thermal conditions. Do not waste your time in trying different polymerase conditions and buffers. Since you already got some success, use the same polymerase and try to go down with the annealing temperature. I suspect it is all about optimizing thermal conditions.

Wish you all success

You need to make sure that the regions you want to PCR, have the exact sequence for primer binding. Otherwise you will get into troubles. If you are using human or mouse genomic DNA, why do not you look for a BAC (bacterial artificial chromosome) containing your sequence of interest. Besides being much smaller than genomic DNA, you have less possible off-target possibilities. We normally make primers 24 nts long and run them at 56C annealing temperature no matter what programmes tell. In the worse of cases we test them in a gradient PCR 50-65C. Good luck

I am for what Mohamed Bessat told you here. Instead of DMSO you can add, as someone pointed here, BSA (0.75 ul of 10 mg/ml BSA per 25 final volume of mixture). Try to use also 100% deionized formamide (0.25 ul/25 ul final volume), this can help you to keep all your primers and DNA template in a single-stranded state. In my experiments all additional stuff are working properly, but it can depend on polymerase or water we use.

Hi Forest, to my experience the betaine additives mentioned by Rocio above helps a lot in case of genomic amplifications. (NH4)2SO4- additives also may be worth to try. Could also try some kits containing additives in order to avoid huge tests of different chemicals at different concentrations.

Good luck

Roland

First of all, go to idtdna.com and go their oligo analyzer tool. Look at primer secondary structure, self annealing, dimer pair formation etc. This can help you determine the quality of your primers.

Second, use gradient PCR which applies a range of temperatures during the reaction. By far, varying the annealing temperature is one of the most important parameters and always the first thing I try when my PCR does not work. Good luck!

PCR optimisation is a endless game.... I wont answer anything unless I know wich organism and the sequence of the primer you used...

Hi, Forest, Does your 1.5%MgCl2 concentration mean: you have 1.5g MgCl2 in your 100ml PCR rxn?

If the genomic DNA is fragmented or sheared that my cause some problems. Regions intact may give products and the regions fragmented may not. You can check the integrity of genomic DNA by pulse field gel electrophoresis.

If there is non specific binding then you will get some product in your gel. But you said that the lanes are blank. Therefore, I guess the problem may be also with primer annealing. You may check the primer quality, if necessary use a different set of primers. GC content of the template DNA may also affect, therefore, can use appropriate kit (Advantage GC 2 Polymerase Mix & PCR Kit from clontech) . Primer concentration and template DNA concentration optimization can also help.

Hi , Forest.The size of fragment is affordable to PCR amplification, so any TAQ DNA Polymerase will amplify, just make sure that the DNA is pure without histones in it. I simple gel to look contamination with protein will help to evaluate. In my experience in PCR, a study of anneling gradient will help you set up faster, but you need to think in to evaluate the anneling time. I think, DMO, NH4SO4, and other denaturant need to use with some care, because they inhibity enzyme activity, so, you need to use a acetylated albumin, to protect. Another litle tips, is never to forget to make a heat shock (5 minutes heat and ice bath immediately, and keep sample in there you can spin down for a minute too.

the amount of dNTP also is important for some sequency region that you want to amplify, do you try to reduce a litle?, Also increase the concentration of Mg2 ou Mn2 (depend of taq).

Also increase the anneling and extension time .

good look.

mario

I suggest to use a nested PCR, you can will use to pair of primers of 15 pb or less, giving you specificity and smaller primers for a more ortodox PCR; I can help you in the primer design

http://en.wikipedia.org/wiki/Nested_polymerase_chain_reaction

The first thing you should do is to verify your PCR anneling gradient temperature and try to increase the extension time. The problam may be with the annealing of your set of primers. Success!

IIn my experience, nested PCR may solve the problem. It will increase the specific template at the first PCR---to increase the probability of amplifying the exact frahment in the second PCR.

In my opinion, the quickest way for you to solve the problem is designing another pairs of primers. There are too many parameters for you to "Play with" in PCR, thus, if you are in a hurry, just ignore the failed primers and order the new ones.

The first thing for successful of PCR amplification is to look for the correct annealing temperature. You can use gradient thermal cycler to screen the good annealing temperature for each primer pairs. Different primer pairs may have different annealing T. Furthermore, you tested for 12 genes, you have to check for the annealing T for each primer pairs using singleplex PCR. Annealing T based on the calculation or given by the manufacturer who synthesize the primers usually doesn't work.

As you said that your primers are good, and amplifying 5 out of 7 promoter and then 4 , so its look like you should increase the time in denaturation cycle coz genomic DNA huge and many a time it will be double stand in some part of gc rich region so if you are having your interest of sequence there then no amplification.

Hi Forest I think you should check the correct annealing temperature for the primer which did not give any band. GC content may also affect the taq pol activity so try some other pol.

Hi Forest,

First you have to check DNA both quantitative and qualitative. If you sure that steps are OK. You should design primer set again using a common used software such as Primer3. 3 degree not so small for a primer set.

Good luck.

Hi Forest,

I think you should do a gradient PCR to check appropriate annealing temperature, and use longer extension time for non-detected samples (90-120s). in my case, I use 100-300 ng gDNA for 20uL reaction. You might take higher DNA amount for your reaction.

Hi Forest,

I think most of the usefull hints were already given previously... However, just one small thing in addition: PCRs on genomic DNA are often tricky, so it is "normal" that some of the PCRs do not work instantly. In most cases this is due to high GC content. If you want to have a fast work around, try the GC melt kit from clontech, it is pretty expensive, but in our hands it saved us a lot of time!

Best

Volker

Hi Forest,

I agree with the comments highlighting the importance of annealing temperature, which should be optimised for each primer pair (ideally with a gradient thermocycler). But also, have you tried to increase Cl2Mg? You can try with 2mM or even 2.5mM. This generally works fine in my hands. Hope this helps. Good luck.

Q: Are you doing a multiplex PCR? If yes, why not try to amplify your segments one by one? Sometimes, multiplexing can be a headache and you may have to go fragment by fragment to be sure that your primers are really working and not interfering with each other.

Hi Forest,

when using your own PCR reaction mix with DMSO which Taq pol brand you use?

I found several times, that for special genomic fragments as promotors, it's best to use high fidelity polymerases. I have been using Expand Long template PCr system (Roche) with buffer no.3 for several years and only for special amplifications. This buffer no.3 contains betaine and DMSO plus nonidet (a detergent) and this works greatly!

So, if with your PCR system (2.5% DMSO, 1.5mM MgCl2) doesn't work the gradient PCR to establish the correct amplification temperature, try this kit, it works nicely.

As other guys said, the annealing temperature is a critical factor for successful PCR. Due to the high percentage of GC in the promoter regions, it is more favorable to use higher annealing Temp (more than 60 degree). An excellent program for calculating the real Tm is "Base Stacking Tm" that you can access it at:

http://www.biophp.org/minitools/melting_temperature/demo.php?formula=Base-Stacking

In many cases, it told you almost actual Tm of your primers or at least a real starting point of Temp for gradient PCR range setup.

DMSO and Betaine, both work well in amplification of Harsh segments of genome.

Other suggestions:

Increase the Extension Time to 70sec/Kb

Increase the initial denaturation if your enzyme allowed (this can improved your result by denaturing all proteins that regularly bind to promoter regions).

Use high-quality DNA polymerases.

Touchdown and Nested PCR (avoid amplifying nonspecific sequences, but you have not any Band in your gel) will not help you. Therefore, do not waste your time.

hello mr. forest,

i little bit understand wih ur problem. In my point of view, you may a few changes in PCR reaction mixture. As you told in BLAST soem of primers are good but experimentally are not exposing their result. So you may do: for 25 microlitre reaction mixture: Your DNA concentartion should be round 60-65 ng (adjust around 2 microlitre volume;), 10 X buffer should be 1X (2.5 microlitre without MgCl2), MgCl2 should be added seperately with 2.5 microlite then both primers of 2 microlitre each (50ng) then dNTPs are 2.5 microlitre with final concentraion of 50 micromole and ultimately addiion of Taq polymerase 0.5 microlitre (3 units), rest volume is maintained by triple distilled water and adjust the PCR machine with your optimum temperature condition and cycles will be around 35 cycles. i hope for the best results.

Hi Forest,

Well I think you have got amply of good suggestion. But to my experience if it is possible try to use some more efficient enzyme like Phusion High fidelity enzyme

http://www.neb.com/nebecomm/products/productm0530.asp

And as some one suggested try to do gradient PCR to see what temperatures are most suitable for you. This enzyme can be used for high GC rich region amplification also please read about this enzyme. I have provided the link above.

Best of luck

Prasoon

Hello Forest

Try to do a nested PCR with two couples of primers, the first PCR with DNA genomic and the first couple of primer and the second with the PCR product with the second couples of primer (inside).

Good luck

Hello,

I have good experience with Takara LA Taq (with GC buffer). If it is still troublesome, you could try to nest your PCR. Make two new primers ~100 bp upstream and downstream from your promoter region, run them first and then use 1 µl of the product as template in your final PCR.

Good luck

I had good results by adding Formamide 5% final, in Mycobacterial DNA regions really diffcult to amplify, but also the sugggestion to prolong the extansion time is useful.

Most likely the GC will be the problems. I often get good results using 5x GC solution from roche. You can try with fast-start taq and its GC rich solution from Roche. Hope it works!

Dear Forest, in my experience adding betaine has solved all the problems with GC rich regions (as the other collegues have suggested you). Moreover I can suggest you to try GoTaq Flexi (Promega) which is a very efficient polymerase. Let us know!

If possible, I suggest that you use BAC clone to replace genomic DNA as the PCR template.

High GC may be important, as others have mentioned. Additives to improve melting or the use of commercial "high GC" buffers may be helpful. You can also try raising the melting temp of the denaturation phase of the PCR to 98 C and/or extending the time of this step to ensure that the DNA is completely melted. To further check your primers, try the In silico PCR function at http://genome.ucsc.edu/cgi-bin/hgGateway. Click on "Tools" and select In silico PCR from the pull-down menu. Select your organism and paste in your primer sequences to verify your expected product.

I am with the others that suggest changes for the high GC content of the fragments, and a polymerase specific for this problem. I also would suggest checking the primers for the non-working fragments and design them with closer melting temperatures, and introduce the changes in the reaction suggested by David.

You may want to try altering thermal cycling conditions. Try standard PCR cycling with 55C annealing and longer incubation times (~ 60 sec for each step).

You may want to try altering thermal cycling conditions. Try standard PCR cycling with 55C annealing and longer incubation times (~ 60 sec for each step).

Hi everyone,

Thanks for all the advice! Trying out all the suggestions will take me some time & hopefully I won't need to actually attempt everything suggested. I wanted to try just using betaine first (one new variable at a time), but neither my lab nor the neighboring ones have any on hand (although I have ordered some). I was able to borrow another lab's Phusion DNA polymerase kit from NEB, which has a buffer that is optimized for amplifying high-GC content sequences (I don't know what it contains). I followed the manufacturer's protocol & ran the PCR for a total of 35 cycles. Only some of the resulting bands were visible, but encouragingly, more bands than I had obtained in the past became visible after adjusting contrast & brightness levels using ImageJ. Based on this, I'm planning to repeat this protocol tomorrow, for 45-48 cycles.

@Karen Posey: I hadn't thought of digesting the DNA beforehand. That makes sense. When you did it, did you just use any enzyme that doesn't cut within your desired sequences, or was there something else more specific?

As this question has generated so many responses, I'll be sure to update further as I try more things. The nested PCR strategy also sounds promising, although I'll wait and see how the next couple of optimization attempts go. Thanks again!

Hi everyone,

Thanks for all the advice! Trying out all the suggestions will take me some time & hopefully I won't need to actually attempt everything suggested. I wanted to try just using betaine first (one new variable at a time), but neither my lab nor the neighboring ones have any on hand (although I have ordered some). I was able to borrow another lab's Phusion DNA polymerase kit from NEB, which has a buffer that is optimized for amplifying high-GC content sequences (I don't know what it contains). I followed the manufacturer's protocol & ran the PCR for a total of 35 cycles. Only some of the resulting bands were visible, but encouragingly, more bands than I had obtained in the past became visible after adjusting contrast & brightness levels using ImageJ. Based on this, I'm planning to repeat this protocol tomorrow, for 45-48 cycles.

@Karen Posey: I hadn't thought of digesting the DNA beforehand. That makes sense. When you did it, did you just use any enzyme that doesn't cut within your desired sequences, or was there something else more specific?

As this question has generated so many responses, I'll be sure to update further as I try more things. The nested PCR strategy also sounds promising, although I'll wait and see how the next couple of optimization attempts go. Thanks again!

BTW, restriction digest of your products with DpnI cuts the template DNA leaving only PCR product. May help also to use cell culture as template rather than purified DNA. In theory, a few cells is sufficient for amplification.and cells have an amazing ability to replicate even the sequences we find to be difficult. Perhaps there exists some factor, in vivio which assist the polymerase with replication that doesn't exist now in most commercial products. But Phusion is amazingly fast. Best of luck.

In addition to using betaine, when I do genomic PCR I like to increase the Tm on my PCR primers and raise the annealing temperature. Unless the DNA is fragmented, genomic DNA can reanneal at higher temperatures than plasmid or cDNA, thus making it more difficult for primers to anneal.

Dear Forest,

You didn't mention G/C contents of any of your primers.

1. First check G/C ratio and then compare both primer pairs i.e. Five pairs which gave amplification and seven pairs which gave no amplification in PCR.

2. try using 2 % or 2.5% MgCl2 with 2% or 2.5% DMSO.

3. Use 100 to 150 ng Genomic DNA. You also didn't mention organism from which you are extracting DNA. The method of DNA extraction?

Anyhow, I hope these points will solve your problem. Good Luck!

Dear Forest,

You didn't mention G/C contents of any of your primers.

1. First check G/C ratio and then compare both primer pairs i.e. Five pairs which gave amplification and seven pairs which gave no amplification in PCR.

2. try using 2 % or 2.5% MgCl2 with 2% or 2.5% DMSO.

3. Use 100 to 150 ng Genomic DNA. You also didn't mention organism from which you are extracting DNA. The method of DNA extraction?

Anyhow, I hope these points will solve your problem. Good Luck!

I would also add that for high complexity template, like genomic DNA, a hot start for the PCR can make a significant difference. Phusion has a hot start version but if you have the standard one just adding the enzyme after the reaction had reached 70-80ºc work just as well.

First check the contents of G/C to calculate times of elongation and play whit a curve of calibration around the temperature calculated.... use the better.

second i agree whit Sarfraz Kiani on the secon passss.

third equalize all the dna or rna to use i preffer 150 to 200 ng but depend the kind of organism and if its tissue, cells or an specific organ tissue...

standarize every point of your protocols

Luck!!!!!

I agree with the purposes brought up here. Betaine is very useful for difficult PCRs and the appropriate G/C content of the primers is vital for good PCR efficiency. Instead of performing a "true hotstart" with a special enzyme you can also leave the Polymerase out of the PCR-Mix, extend the first denaturation step of the PCR to 5 - 7 minutes and add the polymerase afterwards. You can also try to increase the number of cycles up to 40, but the efficiency can also decrease if you do that, you should conduct some tests. In our lab we use the high fidelity PCR kit for complicated PCRs, it mostly works even on single cell level. Good luck.:)

Pls follow the PCR reaction from Taq information that you bought from that company. From my work, i started to PCR reaction followed Gotaq information... they will show the suitable condition for Gotaq... and i never used DMSO...

I would try using a hot start PCR enzyme...usually I use this kind of enzyme when I want to amplify a 1kb-long promoter regions. Try to design other pairs of primers with a conserved region to try to amplify other regions and revise the melting temperatures and so you must revise your protocols. It looks like a PCR optimization problem. Good luck!

I agree with Sarfraz. Also, compare anneling temperature of failed 7 primers with previousely published successful PCR reactions. Extend extension period for about 2 minutes and finally use references to optimize your PCR reactions. Good luck

I would do the same story with BACs.

Hi Ray,

As I see, you already have got lot of good advice. According my observation negative result also can help to find the problem. I prefer to exclude the simple problems before start complicated time consuming and expensive experiments.

I would go as follow; first check the template/primer ration and primer binding. It is very easy; just look the gel picture of the PCR. If you have strong primer band on the end of the gel but no PCR amplification, could mean, that your primer did not aligned to the template. It could mean (among others) that the annealing temperature is not correct. I would make first of all a gradient PCR with the same circumstances. The calculated annealing temperature sometimes does not work on all type of template.

On the other hand if you do not see the rest primers on the end of the gel, it means, that it already bound to the template. It could be nonspecific annealing, even if the primer planning program does not find it. In this case the gradient PCR also can help.

Usually only the template – primer ration is not correct, in most cases not enough primers to anneal on both strands of the same allele. Add more primer and repeat the PCR (you have already made PCR with 5 and 10µM primers but I do not know where they annealed or not). Do not change anything else. If the gel picture of the PCR reaction is the same, no amplification and no rest primer, than most possibly you have nonspecific annealing. Gradient!

If the gradient PCR did not show itself and the rest primers still show a strong band on the end of the gel, you have chance that you have to design a new primer.

A last advice: change always only one parameter one times.

Good luck, Maria Berenyi

Hi Ray, I suggest you to use a hot start enzyme to amplify a 1 KB-long promoter region and try to design a new pair of primers. I usually use a High Fidelity enzyme from Invitrogen. Revise your protocols and try to do a gradient PCR, ok? Good luck and let me know if it worked out!

GC% ratio must be calculated firstly then use taq polymerase

Agree with Zemin ....

If you are having difference in anealing tem between the primers then always use anealing tm of low tm primer. like in your case u can choose 57 or 58 degree centigrade. Use all other parameter as same. Alternatively you can do long PCR starting from higher anealing Tm 60 to 57 deg. In most of the PCR anealing tm optimization play a crucial role for amplification of DNA.

Try HotStart it is very good!

Use primers and enzymes provided by Applied Bio-systems.

You have to know first how are the promoter regions spread in a genome. The genome length matters as the DNA that you normally isolate may be pieces of about 20 kb or more. So it is not possible to expect to all the 12 promoters to be amplified in one go despite the many attempts to optimise the PCR conditions.You may try a BAC or YAC libtrary if you have.

Yeah, I definitely wasn't able to amplify them all in one go. I ended up being able to amplify most of them by doing two touchdown PCRs; one using the Phusion polymerase and the second using the remaining primers and Q5 polymerase.

Siva, can you expand on what you mean by knowing how the promoter regions are spread through a given genome? Thanks.

What I meant was how the promoters are distributed on the genome i.e over how many bp length. If the promoter regions are within a reasonoble length of a genome it may be possible to ampify all of them but if these are spread over differerent chromosomes or over a greater length 9say > 50kb) it may be rather difficult to amplify all at the same time.

Great enzymes for problematic PCRs are Phusion from Thermo Scienitific and PrimeStar from Takara.

In my lab, for promoter regions which are always rich in GC, we order primers with a higher melting temperature, like 70-72 C. We use the Type-it hot-start polymerase (Qiagen) and a mix that contains Q solution (which is in the Type-it kit) and sometimes DMSO. Then instead of a three steps PCR, we only do a two-steps with one denaturation step and one annealing/elongation step. If you need more, I could provide our exact protocol. Good luck!

I have had good success amplifying difficult targets using nested PCR reactions.

There is a way to decrease stringency and still increase specificity. If your problem persist, I could suggest a way to do this.

I had similar problem and the tips here proved to be really useful so here's my contribution. After multiple failures (no amplification at all) I managed to PCR 1300 bp region of interest from mouse genomic DNA by making following alterations to the standard protocol provided with Phusion Hot Start Enzyme:

1. I lowered annealing temperature radically from the one suggested by the protocol online calculator for my primers (64 C) to 58 C.

2. I made EcoRI digestion to the gDNA followed by clean-up using Wizard (PCR) clean-up kit. I used 90 ng of this purified digest for the PCR.

3. I increased the times of preliminary denaturation (98 C) to 2 mins and the actual denaturation (98 C) to 10s. Note that not all polymerases can necessarily take this high temperatures.

4. I increased the extension (72 C) time to 60 sec and final extension (72 C) to 10 mins

5. I increased the number of cycles to 35

The protocol worked nicely even without DMSO but 3% even increased the amplification efficacy. I did not use the standard HF-buffer but the GC-buffer that came with the enzyme. The amount of enzyme I used was quite high (0,5 ul/20ul total volume).

I had the same problem. I was amplifying a gene which had around 75 percent GC content. I tried different things, but didn't work. Best DNA polymerase to amplify high GC content gene is One taq hot start DNA polymerase. Using 10-20 percent GC enhancer (which comes with this enzyme ) in reaction mixture worked for me.