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Problems with PCR, gel electrophoresis, sequencing

Problems with PCR, gel electrophoresis, sequencing - PCR - Polymerase Chain Reaction Forum

Problems with PCR, gel electrophoresis, sequencing - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 02-11-2009, 02:39 AM
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Default Problems with PCR, gel electrophoresis, sequencing



So my first question is this:

I had isolated genomic dna.. ran the taq pcr.. isolated the one band.. purified it from the gel. Before I ran Big Dye PCR, nanodrop showed me 47.2 ng/uL. I ran a big dye pcr rxn with forward sequencing primers...

this PCR tube had: Ready Reaction Premix - 1Ál. Big Dye Sequencing Buffer - 3.5Ál. Primer - 1Ál. Template - 1Ál. Pure Water - 13.5Ál.

I ethanol precipitated this and then sequenced but got absolute crap (biochem professor didn't think it looked like noise but there were some very very low peaks). Any suggestions for what could have gone wrong?? I think maybe the pure water used had a contaminant or maybe I got rid of the pellet during ethanol precipitation?

My second question: I have genomic DNA and after I run taq PCR... I am not able to see any bands! The primers were designed with primer3 and our professor ok'ed them. I even ran a gel at 1.5% agarose to see if I could detect something (our usual is 0.8%).

My taq polymerase reaction reagents look like this:

Taq buffer (10x) - 5Ál
Deoxynucleotides Mix - 2Ál
Upstream primer - 2.5Ál
Downstream primer - 2.5Ál
DNA template - 2Ál
Taq polymerase - 0.5Ál
Nuclease Water - 35.3Ál

Both of the primers used for this have a Tm of like 61 so the standard protocol should be more then fine. Anyone have any suggestions? Currently the genomic DNA has a nanodrop reading of only 18 ng/uL which is kind of low. Could this most likely be my problem? Thanks!
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  #2  
Old 02-25-2009, 02:28 PM
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Default Re: Problems with PCR, gel electrophoresis, sequencing

In my limited experience, I think:

1. Sequencing problem
I can't recall from the top of my head the quantity of components to add, but generally the main factors is the precipation and ethanol wash step. The precipation step with sodium acetate should be extended longer to around half an hour (if you don't do this already). This is to ensure the DNA strands with/without dyes are precipitated to the bottom.

The MOST critical step is the ethanol wash step. After spindown, IMMEDIATELY discard as much ethanol as possible. At this stage, the DNA strands with dyes (the ones you're interested in) are at the bottom, while non-dye bounded nucleotides are floating in the the ethanol. Yet these free-floating nucleotides would be falling quickly back to the bottom of the tube, mixing back with dye-bound nucleotides. Repeat the wash step twice, or do an extra round just to be sure.

If the ethanol wash step is not done correctly, the non-dye bounded nucleotides would interfere with the scanning by the sequencing machine, thereby producing sequence results with low peak, overlappping peaks (noise) and lots of Ns.

2. PCR
Possible things to check:
a. You didn't list the Mg2+. Is it included in the Taq buffer? Typically 1.5 mM of Mg is enough for PCR, but try running an increasing gradient of Mg just to be sure.
b. The DNA extraction process. Are you sure the extraction was carried out correctly, and the DNA source was ok? Check the spectrophotometer values, I think it's the 260 or 260/280 one (look it up in Wikipedia) to make sure that it's DNA content that the nanodrop is measuring. Or try doing another DNA extraction just to be sure.
c. Primers. If they're newly-designed primers, there's no guarantee it would work. Do a blastn on them, what are the range of scores and E-values? High E-values could indicate low specificity, ie your primers bind to multiple sites and non-specific amplification happens. This could be possible, even without any smearing observed.
Also, continue to try temperature gradient at lower temperatures, until you get at least some smearing. There are many ways to calculate primer Tm, and the one stated by the program might not be the best one (try comparing with the one stated on datasheet given by the primer supplier).

Hope this helps.
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  #3  
Old 02-26-2009, 01:19 AM
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Default Re: Problems with PCR, gel electrophoresis, sequencing

My thoughts your predicaments:

1. Sequencing
The main factors are usually the precipitation and ethanol wash step. Try extending the sodium acetate precipitation to 30 minutes (if you haven't already), this would ensure that the dye-bound nucleotides would precipitate to the bottom.

The ethanol wash step is the most CRITICAL step. After spin down, IMMEDIATELY discard as much ethanol as possible. During spindown, the non-dye-bound nucleotides will float around, while the dye-bound nucleotides precipitate to the bottom. If you're too slow to discard the ethanol, the non-dye-bound nucleotides will fall down and mix again with the dye-bound ones. Run an extra wash step if necessary. Poor washing will result in the non-dye-bound ones interfering with the scanning by the sequencer, generating low peaks, noise etc.

The pellet can withstand surprisingly strong amount of force (such as those used while emptying the ethanol violently). Just have faith the pellet is there, though you can't see it.

2. PCR
You did not indicate the Mg2+ component, is it included with the Taq buffer? If it is, likely the default is 1.5 mM Mg. Try running an increasing gradient till you get some faint smears.

Primers: Are they brand new untested primers, or has anyone used them before and gotten results? Run a blastn, are your primers specific for your gene of interest? What are the range of E-values for it?

There are many ways to calculate annealing temperatures; compare the temperature indicated by the primer3 programme and the one supplied by the primer manufacturer? Keep reducing the temperature until you see faint smears.
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  #4  
Old 08-17-2009, 11:52 AM
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Default Re: Problems with PCR, gel electrophoresis, sequencing

Dear everybody
Hi
Hope you are fine
I have a problem, Please help me.
I have done multiplex PCR but my PCR product bands in agarose gel electrophoresis is faint, The first think my DNA have a infected. but at now I have pure DNA. unfortunately don't solve my problem.
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Old 08-17-2009, 08:23 PM
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Default Re: Problems with PCR, gel electrophoresis, sequencing

Maybe you load too little DNA onto your gel if the band is faint? Do you obtain specific PCR product, that is at least the product you wanted?
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Old 08-18-2009, 01:11 AM
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Default Re: Problems with PCR, gel electrophoresis, sequencing

It could also be the case of what kinda dye you are using in your agarose gel electrophoresis. For eg, EtBr usually gives a fainter band than SybrGreen, which in turn, gives a fainter band than GelStar for the same amount of DNA loaded.
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Old 08-20-2009, 03:44 PM
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Default Re: Problems with PCR, gel electrophoresis, sequencing

@biggy1001
just one thing seem to bother me, i think u load too much DNA template for cycle sequencing. do you observed there are a lot of high peak at the beginning of the read? usually this will give very short read. Since we may not be able to see those data especially the electrophorogram, it is a bit hard to deduce from here.

and if that is the case, then the MASSIVE high front reading will make the software adjust all the other peak of interest to a near baseline. so just use the zoom option to drill in those low peak. value of the intensity of the peak should give u clue.

in short-just check those low peak sequence and blast it. does it give u the sequence ur expecting?

2nd part... u didn't include MgCl2?

give us update ya

@ Mtaha
more info is needed to help u!! like how many plex are u doing etc etc maybe a pic of the gel would help
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Old 09-08-2009, 09:03 PM
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Default Re: Problems with PCR, gel electrophoresis, sequencing

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, iNtRONĺs essential technology, the vacuum-dried premix type, maximizes the productĺs stability and convenience in use.
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