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Common problems and answers to PCR instrument
時(shí)間：2018-05-02 來(lái)源：EASTWIN瀏覽次數(shù)：173
Possible reasons:
Low quality of RNA template
* overestimation of mRNA concentration
* there is a shortage of reverse transcriptase inhibitors or reverse transcriptase in the reaction system.
* isotopic phosphorus 32 overdue
* the volume of the reaction is too large and should not exceed 50 mu L
* the most common reason is that your reaction system is the reaction system of PCR rather than the reaction system of RT-PCR.
* related to the total and purity of RNA at the beginning of the reaction
* suggest adding a control RNA to the test
* the content of the first chain reaction product should not exceed 1/10 in the total reaction system when PCR is amplified.
* the use of Oligo (dT) or random primers instead of gene specific primers (GSP) is recommended for first strand synthesis. Because the RNA template has a two stage structure, such as the ring result, it may cause GSP to be unable to be annealed with the template, or the SS II reverse transcriptase can not be effectively extended from the primers.
* target mRNA contains strong transcriptional stop sites, which can be solved by following the following methods:
A. increased the reaction temperature of the first chain to 50.
B. uses random dimers instead of Oligo (dT) to perform the first chain reaction.
* RT negative control was used to detect whether DNA was contaminated by genomic DNA. If the PCR result of RT negative control also shows the same band, DNase I is needed to reprocess the sample.
* in the PCR reaction, nonspecific initial amplification will result in non-specific results. Annealing at temperatures below the primer Tm 2 to 5 C will reduce the production of nonspecific results by reducing the amount of magnesium ions or the amount of DNA.
* due to the different mRNA cutting methods, different RT-PCR primers will result in different results.
* in the PCR reaction system, the content of the first chain product is too high.
Reduce the amount of primers
Optimize the PCR reaction condition / reduce the number of cycles of PCR
* when DNase is used to treat DNA contaminated RNA samples, the oligonucleotide fragments produced will produce non-specific amplification, which will generally be a diffuse background.
* most of the cases are caused by non-specific initiation and elongation due to the low annealing temperature.
* for the long fragment of PCR, it is recommended to dilute the concentration of cDNA in the reaction system to 1:10 (or 1:100-1:200).
In the absence of reverse transcriptase, RNA amplification results were obtained.
* usually due to the presence of trace amounts of DNA in contrast RNA. It is impossible to eliminate all DNA templates because of in vitro transcription. It is suggested that the first chain cDNA be diluted by 1:10, 1:100 and 1:1000 times to eliminate the effects of DNA pollution.
* it is possible to be a band of primer two polymer
* it may be due to the excessive amount of template that PCR results in the formation of high molecular weight DNA colloids. It is suggested that the first strand should be diluted at least 100 times and then expanded for two times.
In addition, the annealing temperature used at two PCR is 5 degrees lower than the Tm value of primers, which can increase the annealing temperature appropriately or start up the heat to improve the specificity.
* with higher thermal stability (up to 50 degrees C)
Having a longer half-life (up to 220 minutes)
No inhibition of PCR
* dry ice transport
* Tdt activity is lower
If ThermoScript is not preserved properly, the activity will decrease rapidly and SS III will be more stable.
GSP is the best way to amplify low abundance transcripts. OligodT primers recommended for high quality RNA and full-length transcripts reverse transcription; random primers were used for reverse transcription of mRNA fragments.
In the first round of PCR, RNA/DNA heterozygote could not undergo normal degeneration.
Objective suggestion
The use of different primers for RT and PCR
Or you need to choose PCR DNA polymerase two step RT-PCR system flexibly.
High sensitivity one - step or two - step RT-PCR system
Two step RT-PCR system with high specificity and proper DNA polymerase
One step RT-PCR system with high fidelity Platinum Taq enzyme
High fidelity two step RT-PCR system containing Pfx Taq enzyme
Long reverse transcription results usually achieve the best results using the two step RT-PCR system.
One step RT-PCR system containing Elongase enzyme
The use of 5 or 3 'RACE reagent requires at least one gene specific primer. When designing primers, you need to pay attention to the following requirements:
* GC content of 50-70% to increase the melting point of primers (Tm).
* 23-28 base lengths to improve primer specificity
* reduce the 3 'terminal GC content and minimize the possibility of primer nonspecific binding.
* join the Hela control
* Low quality RNA templates
* reverse transcription failed. SSII and SSIII are very suitable for the synthesis of long template cDNA.
* the target gene abundance is too low, which can be solved by increasing the number of cycles of PCR. Nested PCR is recommended.
* the target gene is not expressed. It can be used to analyze whether cDNA contains target genes by using two GSPs.
* the target gene is too long and is not suitable for reverse transcription. It is recommended to use Oligo dT in the GeneRacer kit to obtain full length cDNA, using random primers or PCR with the 5 'end of the template as close as possible to GSP.
* cDNA template is a difficult template, which can be solved by optimizing the PCR reaction parameters and reaction system; reducing annealing temperature; using the DMSO of 5-10% to help through the high GC content area; using high fidelity and high extention enzymes for PCR reaction.
RACE PCR heterozygosity or non-specific PCR bands may be due to the following reasons:
* nonspecific binding of GSP to other cDNA results in unrelated products when the target product is amplified.
* the nonspecific combination of GeneRacer primers and cDNA leads to the production of PCR products with GeneRacer primer sequences at one end.
* RNA degradation.
* PCR tube or reagent pollution.
Note: the miscellaneous band is usually because no optimized PCR condition can be added to the negative control.
* RNA degradation after CIP reaction produces a new fracture template with 5 'phosphoric acid, which can be connected to GeneRacer RNA Oligo. Be careful to operate in order to ensure that RNA is not degraded.
* CIP dephosphorylation is incomplete, which can increase the amount of CIP in the reaction or reduce the amount of RNA.
* PCR produces a heterozygosity and is not a real link product. We can optimize PCR with the above recommendation.
At the time of PCR:
* make sure that you do not use excessive initial DNA or high concentration primers, nor do you add excessive Mg++.
* Please make sure that you are using the appropriate annealing temperature
Please make sure that you do not use excessive DNA polymerase
(1) which purification method should be chosen?
Depending on the purpose of the experiment and the length of the primers
(2) why did I order 50nmol, but only 40nmol?
50nmol is the starting amount
(3) how to prepare the storage liquid of 100 mu M?
Volume (mu L) = the number of nmol in the quality inspection report * 10
(4) how to design primers?
* general length 20-30bp;
* at least 50% of GC content;
* avoid primer two - polymer and two - grade structure;
The Tm value of the primer pair should be close.
(5) there is an insertion or deletion of the primer sequence.
* use upstream and downstream primers to test multiple clones.
* Please choose the right method of purification.
(6) there is no result in PCR?
* Please check the correctness of the primer design;
* Please check whether the OD readings are correct;
* make a positive control and a negative control.
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In order to improve the accuracy of the experiment and reduce the problems and risks in these experiments, it is very important to choose a suitable and reliable instrument to carry out experimental operation. Suzhou Dongsheng Xingye Science Instrument Co., Ltd., the electronic ice box, ETC811PCR instrument and the follow-up instrument of PCR, the blue light cutting instrument for your perfect experiment escort.
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eICE-4
電子冰盒 Electronic Ice Box  |
ETC811
基因擴(kuò)增儀 Thermal Cycler  |
eBL-100
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