1. The yield of cDNA is very low
possible reason:
*RNA template quality is low
*Overestimate the mRNA concentration
*There is insufficient amount of reverse transcriptase inhibitor or reverse transcriptase in the reaction system
*Isotopic phosphorus 32 expired
*The reaction volume is too large and should not exceed 50μl
2. The amplified product has no bands or bands in the electrophoresis analysis.
* The most common reason is that your reaction system is a PCR reaction system rather than an RT-PCR reaction system.
*Related to the total amount and purity of RNA at the start of the reaction
*Recommended to add control RNA to the test
* When the reaction product of the first chain is subjected to PCR amplification, the content in the total reaction system should not exceed 1/10.
* It is recommended to use Oligo (dT) or random primers instead of gene-specific primers (GSP) for first strand synthesis. Due to the secondary structure of the RNA template, such as a circular result, it is possible that the GSP cannot anneal to the template; or the SSII reverse transcriptase cannot be effectively extended from this primer.
* The target mRNA contains a strong transcriptional stop site, which can be solved by the following methods:
a. Increase the reaction temperature of the first chain to 50 °C.
b. Use a random hexamer instead of Oligo (dT) for the first strand reaction.
3. Produce non-specific bands
* Use RT negative controls to detect contamination by genomic DNA. If the PCR results for the RT negative control also show the same band, then the sample needs to be reprocessed with DNase I.
* In a PCR reaction, non-specific initial amplification will result in non-specific results. Annealing at temperatures below 2 to 5 ° C of the primer Tm reduces the amount of magnesium ions or DNA of interest will reduce the production of non-specific results.
* Due to differences in the way the mRNA is cleaved, different RT-PCR results will result depending on the choice of primers.
4. Produce a smear strip
* The content of the first chain product is too high in the PCR reaction system
* Reduce the amount of primers
*Optimize PCR reaction conditions / reduce the number of PCR cycles
* When DNA samples contaminated with DNA are treated with DNase, the resulting oligonucleotide fragments will produce non-specific amplification and will generally appear as a diffuse background.
5. Produce a large molecular weight dispersion strip
* In most cases, due to non-specific initiation and extension due to low annealing temperatures
* For long-segment PCR, it is recommended to dilute the concentration of cDNA in the reaction system to 1:10 (or 1:100-1:200)
6. In the absence of reverse transcriptase, the control RNA obtains amplification results
* Usually due to the presence of trace amounts of DNA in the control RNA. It is not possible to eliminate all DNA templates due to in vitro transcription. It is recommended to dilute the first strand cDNA by 1:10, 1:100, 1:1000 to eliminate the effects of DNA contamination.
* There may be a band of primer dimers
7. The amplified product is retained in the well
* It is possible that the PCR result resulted in a high molecular weight DNA gel due to the high amount of template. It is recommended to dilute the first strand result by at least 100 fold before performing secondary amplification.
* In addition, if the annealing temperature used in the secondary PCR is 5 ° C lower than the Tm value of the primer, the annealing temperature may be appropriately increased or a hot start may be performed to increase the specificity.
8. What is the difference between SSIII and SSII?
* has higher thermal stability (up to 50 ° C)
* Has a longer half-life (up to 220 minutes)
*No inhibition on PCR
*dry ice transport
*Tdt activity is lower
9. Why do people prefer to use SSIII instead of ThermoScript?
If the ThermoScript is not properly stored, it will cause the activity to decrease quickly, and the SSIII will be more stable.
10. Why use gene-specific primers (GSP)?
GSP is best for amplifying low abundance transcripts. OligodT primers are recommended for reverse transcription of high quality RNA and full length transcripts; random primers are used for reverse transcription of mRNA fragments.
11. When do I need to use RNase H?
When the RNA/DNA hybrid is not normally denatured in the first round of PCR
12. Choose a different system for different purposes:
Purpose suggestion
RT and PCR use different primers
Or need flexible selection of PCR DNA polymerase two-step RT-PCR system
High sensitivity one-step or two-step RT-PCR system
High-specificity two-step RT-PCR system with appropriate DNA polymerase
Or a one-step RT-PCR system with high fidelity Platinum Taq enzyme
High fidelity two-step RT-PCR system with Pfx Taq enzyme
Long reverse transcription results usually use a two-step RT-PCR system for optimal results
One-step RT-PCR system containing Elongase enzyme
Second, Generacer
1. How do you design a gene-specific primer (GSP) for the Generacer kit?
At least one gene-specific primer is required to use the 5' or 3' RACE reagent. You need to pay attention to the following requirements when designing primers:
*50-70% GC content to increase primer melting point (Tm)
*23-28 base lengths to increase primer specificity
* Reduce the GC content at the 3' end to minimize the possibility of non-specific binding of primers
2. Why can't I get the RACE product?
*Join the Hela control
*Low quality RNA template
* Reverse transcription failure, SSII and SSIII are very suitable for the synthesis of long template cDNA
*The abundance of the target gene is too low, which can be solved by increasing the number of PCR cycles. It is recommended to use nested PCR.
*The gene of interest is not expressed, and it is possible to analyze whether the cDNA contains the gene of interest by using two GSPs.
* The gene of interest is too long to be reverse transcribed. It is recommended to use Oligo dT in the GeneRacer kit to obtain full-length cDNA, and PCR is performed using random primers or GSP as close as possible to the 5' end of the template.
*The cDNA template is a difficult template and can be solved by optimizing the PCR reaction parameters and reaction system; lowering the annealing temperature; using 5-10% DMSO to help pass the high GC content region; using high fidelity and high elongation enzymes PCR reaction.
3. RACE PCR results have miscellaneous bands
RACE PCR bands or non-specific PCR bands may be due to the following reasons:
* Non-specific binding of GSP to other cDNAs results in irrelevant products when amplifying the desired product.
* Non-specific binding of the GeneRacer primer to cDNA results in the production of a PCR product with a GeneRacer primer sequence at one end.
* RNA degradation.
* PCR tube or reagent contamination.
Note: The band is generally determined because no PCR conditions are optimized and a negative control can be added.
4. Unable to get full length 5'RACE PCR product
* RNA degradation after CIP reaction produces a new cleavage template with 5' phosphate that can be linked to GeneRacer RNA Oligo. Be careful to ensure that the RNA is not degraded.
*CIP dephosphorylation is incomplete and can increase the amount of CIP in the reaction or reduce the amount of RNA.
*PCR produces a band, not a true ligation product, and the above recommendations can be used to optimize PCR.
Second, Generacer
1. How do you design a gene-specific primer (GSP) for the Generacer kit?
At least one gene-specific primer is required to use the 5' or 3' RACE reagent. You need to pay attention to the following requirements when designing primers:
*50-70% GC content to increase primer melting point (Tm)
*23-28 base lengths to increase primer specificity
* Reduce the GC content at the 3' end to minimize the possibility of non-specific binding of primers
2. Why can't I get the RACE product?
*Join the Hela control
*Low quality RNA template
* Reverse transcription failure, SSII and SSIII are very suitable for the synthesis of long template cDNA
*The abundance of the target gene is too low, which can be solved by increasing the number of PCR cycles. It is recommended to use nested PCR.
*The gene of interest is not expressed, and it is possible to analyze whether the cDNA contains the gene of interest by using two GSPs.
* The gene of interest is too long to be reverse transcribed. It is recommended to use Oligo dT in the GeneRacer kit to obtain full-length cDNA, and PCR is performed using random primers or GSP as close as possible to the 5' end of the template.
*The cDNA template is a difficult template and can be solved by optimizing the PCR reaction parameters and reaction system; lowering the annealing temperature; using 5-10% DMSO to help pass the high GC content region; using high fidelity and high elongation enzymes PCR reaction.
3. RACE PCR results have miscellaneous bands
RACE PCR bands or non-specific PCR bands may be due to the following reasons:
* Non-specific binding of GSP to other cDNAs results in irrelevant products when amplifying the desired product.
* Non-specific binding of the GeneRacer primer to cDNA results in the production of a PCR product with a GeneRacer primer sequence at one end.
* RNA degradation.
* PCR tube or reagent contamination.
Note: The band is generally determined because no PCR conditions are optimized and a negative control can be added.
4. Unable to get full length 5'RACE PCR product
* RNA degradation after CIP reaction produces a new cleavage template with 5' phosphate that can be linked to GeneRacer RNA Oligo. Be careful to ensure that the RNA is not degraded.
*CIP dephosphorylation is incomplete and can increase the amount of CIP in the reaction or reduce the amount of RNA.
*PCR produces a band, not a true ligation product, and the above recommendations can be used to optimize PCR.
Third, PCR
When performing PCR:
* Make sure you are not using excessive amounts of starting DNA or too high concentrations of primers, or adding excessive amounts of Mg++
*Please make sure you use the proper annealing temperature
*Please make sure you are not using excessive DNA polymerase
Fourth, the primer
1. Which purification method should I choose?
Depending on the purpose of the experiment and the length of the primer
2. Why did I order 50nmol, but I only received 40nmol?
50nmol is the starting amount
3. How to prepare a 100μM stock solution?
Volume (μl) = number of nmol on the quality inspection report × 10
4. How to design primers?
*General length 20-30bp;
* at least 50% GC content;
* Avoid primer dimers and secondary structures;
* The Tm value of the primer pair should be close.
5. Is the primer sequence inserted or deleted?
* Use the upstream and downstream primers to measure several clones.
*Please choose the correct purification method.
6. PCR has no results?
*Please check if the primer design is correct;
*Please check if the OD reading is correct;
* Make a positive control and a negative control
1. Considering the limitation of storage space and storage cost, as well as the fact that video recording in public places such as residential areas is a prerequisite for multi slot and a certain degree of clarity, video recording is generally deleted once a week or so. 15 days for general entertainment venues, 26 days for financial industry, and 3 months to half a year for banks, depending on the current venue regulations
2. The length of the storage time of the monitoring video is generally related to the following points: the capacity of the host's hard disk. Generally, a camera needs about 0.3G-0.5G of hard disk capacity for 24 hours of continuous video recording. Different systems have different parameters, the minimum is 0.15G/24 hours;
3. The number of cameras. The more cameras, the more capacity required. Also, the selection of the resolution and picture quality of the recorded picture will also affect the capacity required for video storage;
4. The video recording mode will also affect the space required for storage. The video recording mode is generally divided into [Mobile Video Recording] and [Continuous Video Recording]. The former means that the host starts to record when a moving object passes through the monitoring range, and does not record at other times. This can reduce the storage space required for video recording; On the contrary, continuous video recording is 24-hour continuous video recording.
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