optmizing PCR

[fatihatul]

Hi,

I'm doing optimizing PCR. But still did get the band either. i've tried to increase the annealing temperature from -5deg to +5deg. However, it only success to one product out of 5 genes being tested. what should i do to get the band..

[Aga]

Can you give us some more specific information? What reagents concentration do you add, what is the length of your amplified DNA sequence and what temperature pattern do you use in your PCR cycles?

Are you sure your template is ok (well purified, not degraded, enough concentration of DNA) and the primers are well designed? Have you used positive control? Have you tried this protocol before or is it the first time?

[gs_107]

Originally Posted by fatihatul

Hi,

I'm doing optimizing PCR. But still did get the band either. i've tried to increase the annealing temperature from -5deg to +5deg. However, it only success to one product out of 5 genes being tested. what should i do to get the band..

Did U try a Magnesium optimization protocol with different concentrations of Mg? It helped me a few times in the past from problems of getting no band to a huge smear all along the lane. I hope it works our for you !!!!!!

[fatihatul]

hi,

Dear Aga. I'm using 5 primers to detect 5 resistance genes. Those primers were taken from the journal, which i've found may helpful for my research. But, the journal they mentioned they using multiplex PCR, but instead of that i'm using a single (normal) PCR. Will it make lots of difference? Here are the length of amplified sequence i'm looking

SIM- 570bp
GIM- 477bp
IMP- 188bp
SPM- 271bp
VIM- 390bp

By the way, the plasmid that i'm using has a varies length of from 2kb to 200kb.

Below are the recipe i'm using for 50uL

Water- 42uL
Buffer (1X)- 5uL
dNTPS- 1uL
Primers- 0.5uL
Template DNA- 0.5uL
Taq DNA polymerase- 0.5uL

Reaction condition

Initial 94deg 1min
Denaturation 94deg 15sec
Tm varies 30sec
Extension 72deg 30sec
Final extension 72 deg 10min


The Tm varies because the given by the manufacturer and its a bit low from suggested by the journal. The Tm

VIM- 45.25deg
IMP- 43.75 deg
SIM- 46.35deg
SPM- 45.65 deg
GIM- 45.65deg

I've done gradient tempt +/- 5 deg and only works on SPM products and others didn't show a good band (got several bands and primer dimers).

the template is in good condition cause i'm doing plasmid extraction using kit for my plasmid profiling.

[Newscience]

Factors that affect PCR reactions include the concentration of MgCl2 and Taq DNA polymerase, type of Taq DNA polymerase, primer GC ratio, primer Tm, concentration of 10x reaction buffer and primer, and PCR reaction time. As mentioned, the PCR reaction can be changed by various conditions. Therefore, when amplifying a specific gene for the first time, the best PCR conditions can be found by a process of performing PCR many times and modifying the PCR conditions numerous times. Only a few conditions, from the listed conditions above, can be adjusted, such as DNA polymerase type, concentration of MgCl2 and primer, appropriate primer synthesis, and PCR reaction time. However, in order to find out the appropriate PCR conditions, PCR should be performed many times. In other words, a better solution is needed to find the best PCR conditions without having to perform PCR numerous times. To this end, iNtRON has developed the PCR Starter Kit (MS-2 Type), which provides various PCR components in a vacuum-dried premix type.

The PCR Starter Kit (MS-2 Type) consists of 32 different PCR components and will provide 32 different PCR results by one-time PCR so that the customer can control and change the PCR conditions. This new concept product, which overcomes the existing idea that PCR premixes cannot be controlled, provides the desired result in a short time by testing under various conditions, because laborious PCR set-up, in which all buffers have to be mixed individually in order to change the PCR condition, is not required. Also, iNtRON’s essential technology, the vacuum-dried premix type, maximizes the product’s stability and convenience in use.

[fatihatul]

thanx for the suggestion

[berhina27@yahoo.com]

Hi i am berhane teklehaimanot from an African country called Eritrea and i am a masters degree student in the school of biotechnology in jiangnan university(CHINA). I just registered here 'cause i just wanted to gain some knowlege from anybody in this molecular biology forum

[Aga]

Hi fatihatul. I was wondering whether your polymerase buffer already contins Mg2+ or not, cause you haven't mentioned adding this component. Some buffers contain some Mg ions but usually these are low concentrations (i.e. 1mM).

Giving the PCR mixture composition you gave only the amounts of reagents not the concentrations. Because you are doing PCR at 50ul I started to think that the concentration of your reagents may be too low.

Take into consideration polymerase first. Is its concentration 1U/ul? (Some polymerases are 5U/ul.) If you add 0.5U per 50ul reaction it may be not enough and the concentration of the product obtained may be too low to be visualized on the gel.

Check magnesium in your buffer and try to check higher concentrations. From my experience I can say that with some combinations of primers and templates PCR works optimally with Mg2+ concentrations as high as 4-5mM.

It may be similar with other components of your reaction.
I usually work with 20ul PCR volume and I add 2ul of template DNA (10 - 20ng), 0.5 - 0.7ul of each primer (concentration 5mM), 1-4mM Mg2+, 0.5U Taq, dNTP depending on magnesium concentration and 2ul of 10x buffer. I doesn't mean your PCR will work in similar conditions but try some options.

The fact that you do single PCR and not multiplex is actually very good, cause you are able to optimize PCR conditions for each reaction.
Remember one thing - relying fully on other works doesn't always bring satisfactory results. Different lab, different equipment, different people. Only once did I come across something (a method) that was actually impossible to be repeated according to the descriptions in the article.

I hope you solve your problem. Good luck

[ramnivas]

I think you should increase the range of your temperature gradient from present condition and you should also try PCR with Phusion polymerase enzyme, it may give you better results. Because Phusion is more specific than Taq.