Electrophoretic detection time of PCR products
Generally, it is within 48 hours, and some are best detected on the same day. After more than 48 hours, the band irregularity disappears.
False negative, there is no amplification band PCR reaction in the key link of 1 template nucleic acid preparation, 2 primer quality and specificity, 3 enzyme quality and 4 PCR cycle conditions. The reason for finding the problem should also be analyzed and researched on the above links.
Template: 1 template contains heteroprotein, 2 template contains Taq enzyme inhibitor, protein in 3 template is not digested, especially histone in chromosome, 4 is excessively lost when extracting template, or inhaled phenol. 5 template nucleic acid degeneration is not complete. When the quality of the enzyme and the primer is good, there is no amplification band, and it is very likely that the sample is digested, and the template nucleic acid extraction process is out of order. Therefore, an effective and stable digestion treatment solution should be prepared, and the procedure should be fixed and should not be changed at will. .
Enzyme inactivation: new enzymes need to be replaced, or both old and new enzymes should be used simultaneously to analyze whether false negatives are caused by loss or insufficient enzyme activity. It should be noted that sometimes Taq enzyme or ethidium bromide is forgotten.
Primers: Primer quality, primer concentration, and the concentration of the two primers are symmetrical, which is a common cause of PCR failure or unsatisfactory expansion of the band. Some batches have problems with the quality of primer synthesis. Two primers have a high concentration and a low concentration, resulting in low-efficiency asymmetric amplification. The countermeasures are: 1 Select a good primer synthesis unit. 2 The concentration of the primer should not only look at the OD value, but also pay attention to the primer solution for agarose gel electrophoresis. There must be a primer band, and the brightness of the two primer bands should be roughly the same, such as a primer with a band and a primer without Strips, PCR may fail at this time, and should be resolved in consultation with the primer synthesis unit. If one primer has high brightness and one has low brightness, balance the concentration when diluting the primer. 3 Primers should be stored in high concentration and small amount to prevent multiple freeze-thaw cycles or long-term storage of refrigerators, resulting in failure of primer degradation. 4 Primer design is not reasonable, such as the length of the primer is not enough, the formation of dimers between the primers.
Mg2+ concentration: Mg2+ ion concentration has a great influence on PCR amplification efficiency. Too high concentration can reduce the specificity of PCR amplification. If the concentration is too low, it will affect the PCR amplification yield and even the PCR amplification will not be amplified.
Change in reaction volume: The volume used for PCR amplification is usually 20 ul, 30 ul, 50 ul. Or 100ul, the application of large volume for PCR amplification, is set according to the different purposes of scientific research and clinical testing. After making a small volume such as 20ul, and then making a large volume, it is necessary to mold the condition, otherwise it is easy to fail.
Physical reasons: Denaturation is very important for PCR amplification, such as low denaturation temperature, short denaturation time, and highly likely false negatives; annealing temperature is too low, which can cause non-specific amplification and reduce specific amplification efficiency. High-intensity primers bind to the template to reduce PCR amplification efficiency. It is sometimes necessary to use a standard thermometer to detect denaturation, annealing and extension temperatures in the instrument or water bath, which is one of the reasons for PCR failure.
Appearing tapes or sheet tapes or carpet-like tapes sometimes appear in the form of sheet-like tows or tapes for PCR amplification. The reason is often due to the excessive amount of enzyme or the poor quality of the enzyme, the dNTP concentration is too high, the Mg2+ concentration is too high, the annealing temperature is too low, and the number of cycles is too high. The countermeasures are as follows: 1 reduce the amount of enzyme, or exchange enzymes from another source. 2 reduce the concentration of dNTP. 3 appropriately reduce the concentration of Mg2+. 4 increase the amount of template, reduce the number of cycles.
What are the optimal conditions for cloning PCR products?
Optimal insert: The vector is determined experimentally. 1:1 (insert: carrier) is often the best ratio, and the molar ratio is 1:8 or 8:1. The ratio range should be determined. The ligation was carried out with 5 ul of 2X ligation solution, 50 ng of plasmid DNA, 1 Weiss unit of T4 ligase, and a total of 10 ul of the insert. Incubate for 1 hour at room temperature or overnight at 4oC. At these two temperatures, the carrier lacking the T-bulk will self-ligate and produce a blue spot. Incubation at room temperature for 1 hour can meet most of the cloning requirements. To improve the efficiency of the connection, 4oC is required overnight.
Does the PCR product need to be purified by gel?
For example, the gel analysis of the amplified product has only one band and does not require gel purification. If other bands are visible, it may be a dimer that accumulates a large number of primers. The small number of primer dimers is also high in moles, which results in a high proportion of clones with primer dimers, rather than the insert of interest. For this purpose, gel purification is required prior to cloning.
What kind of control experiment is needed if the target fragment is not recovered?
A) Coating untransformed competent cells. If there are colonies, it indicates that the ampicillin has failed, or the plasmid with the ampicillin-resistant type is contaminated, or the colony that produces the ampicillin-resistant type.
B) Transformation of the intact plasmid, calculation of the number of colony growth, and determination of transformation efficiency. For example, 1 ug/ul plasmid was diluted 1:100 and 1 ul was used for 100 ul of competent cell transformation. After diluting to 1000 ul with SOC, plate with 100 ul. Incubate overnight to produce 1000 colonies. Conversion rate: Total number of colonies produced / total amount of plated DNA. The total amount of plating DNA is the amount used for the conversion reaction divided by the dilution factor. Specifically, 10 ng of DNA was used for transformation, and 10 ng of DNA was diluted with SOC to 1000 u, and plated with 1/10 to share 1 ng of DNA. Conversion rate: 1000 clone X10 (3th power) ng / plating 1 ng DNA ug = 10 (6th power) cfu / ug transformation pGEM-T application 10 (8th power) cfu / ug competent cells such as no colonies or There are few colonies, and the conversion rate of competent cells is too low.
C) If a positive control of pGEM-T, or a PCR product, is used to generate >20-40 plaques (using a specified step 10 (8th power) cfu/ug competent cells), indicating that the vector has lost T. It may be that the ligase contaminates the nuclease. T4 DNA ligase (M1801, M1804, M1794) has good quality standards without nuclease contamination and is not replaced with other sources of T4 DNA ligase.
D) Using pGEM-T or pGEM-T Easy vector, connect pGEM-T positive control, transform high frequency competent cells (10 (8th power) cfu / ug), according to the specified experimental procedure, can get 100 colonies, 60% of them should be white spots, such as producing >20-40 blue spots, no colonies or few colonies, and there is a problem with the connection.
The results of the control experiment were good, but the target fragment was not recovered. What was wrong with the experiment?
A) The connection is incubated for 1 hour at room temperature to satisfy most clones. To improve efficiency, 4oC is required overnight.
B) The insert is contaminated, causing the 3'-T to be deleted, or inhibiting the junction, inhibiting transformation. To do this, the insert was mixed with the pGEM-T positive control and ligated. If the number of colonies in the control is reduced, the insert is purified or re-prepared. If a large amount of blue spots are produced, the insert is contaminated with nuclease, and the pGEM-T or pGEM-T Easy vector 3'-T is deleted.
C) The insert is not suitable for connection. Gel-purified inserts sometimes occur due to excessive UV exposure. UV over-irradiation produces pyrimidine dimers that are not conducive to ligation and the DNA must be repurified.
D) The amplified product of the thermostable DNA polymerase with repair function has no A at the end, and the latter is required for pGEM-T or pGEM-T Easy vector cloning. Add Taq DNA polymerase and nucleotides to add A to the end. For details, check the pGEM-T pGEM-T Easy carrier technical data (TM042).
E) Highly repetitive sequences may be unstable, causing deletions and rearrangements in amplification. If the insert is found to be highly frequent in deletion and rearrangement, recombinant defective E. coli strains such as SURE cells are required.
Generally, it is within 48 hours, and some are best detected on the same day. After more than 48 hours, the band irregularity disappears.
False negative, there is no amplification band PCR reaction in the key link of 1 template nucleic acid preparation, 2 primer quality and specificity, 3 enzyme quality and 4 PCR cycle conditions. The reason for finding the problem should also be analyzed and researched on the above links.
Template: 1 template contains heteroprotein, 2 template contains Taq enzyme inhibitor, protein in 3 template is not digested, especially histone in chromosome, 4 is excessively lost when extracting template, or inhaled phenol. 5 template nucleic acid degeneration is not complete. When the quality of the enzyme and the primer is good, there is no amplification band, and it is very likely that the sample is digested, and the template nucleic acid extraction process is out of order. Therefore, an effective and stable digestion treatment solution should be prepared, and the procedure should be fixed and should not be changed at will. .
Enzyme inactivation: new enzymes need to be replaced, or both old and new enzymes should be used simultaneously to analyze whether false negatives are caused by loss or insufficient enzyme activity. It should be noted that sometimes Taq enzyme or ethidium bromide is forgotten.
Primers: Primer quality, primer concentration, and the concentration of the two primers are symmetrical, which is a common cause of PCR failure or unsatisfactory expansion of the band. Some batches have problems with the quality of primer synthesis. Two primers have a high concentration and a low concentration, resulting in low-efficiency asymmetric amplification. The countermeasures are: 1 Select a good primer synthesis unit. 2 The concentration of the primer should not only look at the OD value, but also pay attention to the primer solution for agarose gel electrophoresis. There must be a primer band, and the brightness of the two primer bands should be roughly the same, such as a primer with a band and a primer without Strips, PCR may fail at this time, and should be resolved in consultation with the primer synthesis unit. If one primer has high brightness and one has low brightness, balance the concentration when diluting the primer. 3 Primers should be stored in high concentration and small amount to prevent multiple freeze-thaw cycles or long-term storage of refrigerators, resulting in failure of primer degradation. 4 Primer design is not reasonable, such as the length of the primer is not enough, the formation of dimers between the primers.
Mg2+ concentration: Mg2+ ion concentration has a great influence on PCR amplification efficiency. Too high concentration can reduce the specificity of PCR amplification. If the concentration is too low, it will affect the PCR amplification yield and even the PCR amplification will not be amplified.
Change in reaction volume: The volume used for PCR amplification is usually 20 ul, 30 ul, 50 ul. Or 100ul, the application of large volume for PCR amplification, is set according to the different purposes of scientific research and clinical testing. After making a small volume such as 20ul, and then making a large volume, it is necessary to mold the condition, otherwise it is easy to fail.
Physical reasons: Denaturation is very important for PCR amplification, such as low denaturation temperature, short denaturation time, and highly likely false negatives; annealing temperature is too low, which can cause non-specific amplification and reduce specific amplification efficiency. High-intensity primers bind to the template to reduce PCR amplification efficiency. It is sometimes necessary to use a standard thermometer to detect denaturation, annealing and extension temperatures in the instrument or water bath, which is one of the reasons for PCR failure.
Target sequence variation: If the target sequence is mutated or deleted, it affects the specific binding of the primer to the template, or the primer and the template lose complementary sequence due to a certain deletion of the target sequence, and the PCR amplification is not successful.
The PCR-amplified bands that appear as false positives are consistent with the target target sequence bands, and sometimes the bands are more tidy and the brightness is higher.
Primer design is not suitable: the selected amplified sequence has homology with the non-target amplified sequence, so when PCR amplification is performed, the amplified PCR product is a non-target sequence. The target sequence is too short or the primer is too short to be prone to false positives. Primers need to be redesigned.
Cross-contamination of target sequences or amplification products: There are two reasons for this contamination: one is cross-contamination of the entire genome or large fragments, leading to false positives. This false positive can be solved by the following methods: 1 Care should be taken to keep the target sequence inhaled into the sample gun or spilled out of the centrifuge tube. 2 All reagents or equipment should be autoclaved except for enzymes and substances that cannot withstand high temperatures. The centrifuge tube and sample tip used should be used at one time. 3 If necessary, the reaction tube and reagents are irradiated with ultraviolet light to destroy the nucleic acid present before the specimen is added. The second is the contamination of small fragments of nucleic acids in the air. These small fragments are shorter than the target sequences, but have some homology. The splicing can be spliced ​​to each other, and after complementing the primers, the PCR product can be amplified, resulting in the generation of false positives, which can be alleviated or eliminated by nested PCR.
The PCR-amplified bands that appear as false positives are consistent with the target target sequence bands, and sometimes the bands are more tidy and the brightness is higher.
Primer design is not suitable: the selected amplified sequence has homology with the non-target amplified sequence, so when PCR amplification is performed, the amplified PCR product is a non-target sequence. The target sequence is too short or the primer is too short to be prone to false positives. Primers need to be redesigned.
Cross-contamination of target sequences or amplification products: There are two reasons for this contamination: one is cross-contamination of the entire genome or large fragments, leading to false positives. This false positive can be solved by the following methods: 1 Care should be taken to keep the target sequence inhaled into the sample gun or spilled out of the centrifuge tube. 2 All reagents or equipment should be autoclaved except for enzymes and substances that cannot withstand high temperatures. The centrifuge tube and sample tip used should be used at one time. 3 If necessary, the reaction tube and reagents are irradiated with ultraviolet light to destroy the nucleic acid present before the specimen is added. The second is the contamination of small fragments of nucleic acids in the air. These small fragments are shorter than the target sequences, but have some homology. The splicing can be spliced ​​to each other, and after complementing the primers, the PCR product can be amplified, resulting in the generation of false positives, which can be alleviated or eliminated by nested PCR.
The band appearing after the non-specific amplification band PCR amplification is inconsistent with the expected size, either large or small, or both the specific amplification band and the non-specific amplification band. The emergence of non-specific bands is due to the fact that the primers are not completely complementary to the target sequence or the primers polymerize to form a dimer. Second, the Mg2+ ion concentration is too high, the annealing temperature is too low, and the number of PCR cycles is too high. The second is the quality and quantity of the enzyme. Sometimes the enzymes of some sources are prone to non-specific bands and the enzymes of another source do not. Excessive amounts of enzymes sometimes lead to non-specific amplification. The countermeasures are: 1 Redesign the primer if necessary. 2 Reduce the amount of enzyme or exchange the enzyme from another source. 3 reduce the amount of primers, increase the amount of template, and reduce the number of cycles. 4 Appropriately increase the annealing temperature or use two temperature point method (denaturation at 93 °C, annealing and extension at around 65 °C).
Appearing tapes or sheet tapes or carpet-like tapes sometimes appear in the form of sheet-like tows or tapes for PCR amplification. The reason is often due to the excessive amount of enzyme or the poor quality of the enzyme, the dNTP concentration is too high, the Mg2+ concentration is too high, the annealing temperature is too low, and the number of cycles is too high. The countermeasures are as follows: 1 reduce the amount of enzyme, or exchange enzymes from another source. 2 reduce the concentration of dNTP. 3 appropriately reduce the concentration of Mg2+. 4 increase the amount of template, reduce the number of cycles.
What are the optimal conditions for cloning PCR products?
Optimal insert: The vector is determined experimentally. 1:1 (insert: carrier) is often the best ratio, and the molar ratio is 1:8 or 8:1. The ratio range should be determined. The ligation was carried out with 5 ul of 2X ligation solution, 50 ng of plasmid DNA, 1 Weiss unit of T4 ligase, and a total of 10 ul of the insert. Incubate for 1 hour at room temperature or overnight at 4oC. At these two temperatures, the carrier lacking the T-bulk will self-ligate and produce a blue spot. Incubation at room temperature for 1 hour can meet most of the cloning requirements. To improve the efficiency of the connection, 4oC is required overnight.
Does the PCR product need to be purified by gel?
For example, the gel analysis of the amplified product has only one band and does not require gel purification. If other bands are visible, it may be a dimer that accumulates a large number of primers. The small number of primer dimers is also high in moles, which results in a high proportion of clones with primer dimers, rather than the insert of interest. For this purpose, gel purification is required prior to cloning.
What kind of control experiment is needed if the target fragment is not recovered?
A) Coating untransformed competent cells. If there are colonies, it indicates that the ampicillin has failed, or the plasmid with the ampicillin-resistant type is contaminated, or the colony that produces the ampicillin-resistant type.
B) Transformation of the intact plasmid, calculation of the number of colony growth, and determination of transformation efficiency. For example, 1 ug/ul plasmid was diluted 1:100 and 1 ul was used for 100 ul of competent cell transformation. After diluting to 1000 ul with SOC, plate with 100 ul. Incubate overnight to produce 1000 colonies. Conversion rate: Total number of colonies produced / total amount of plated DNA. The total amount of plating DNA is the amount used for the conversion reaction divided by the dilution factor. Specifically, 10 ng of DNA was used for transformation, and 10 ng of DNA was diluted with SOC to 1000 u, and plated with 1/10 to share 1 ng of DNA. Conversion rate: 1000 clone X10 (3th power) ng / plating 1 ng DNA ug = 10 (6th power) cfu / ug transformation pGEM-T application 10 (8th power) cfu / ug competent cells such as no colonies or There are few colonies, and the conversion rate of competent cells is too low.
C) If a positive control of pGEM-T, or a PCR product, is used to generate >20-40 plaques (using a specified step 10 (8th power) cfu/ug competent cells), indicating that the vector has lost T. It may be that the ligase contaminates the nuclease. T4 DNA ligase (M1801, M1804, M1794) has good quality standards without nuclease contamination and is not replaced with other sources of T4 DNA ligase.
D) Using pGEM-T or pGEM-T Easy vector, connect pGEM-T positive control, transform high frequency competent cells (10 (8th power) cfu / ug), according to the specified experimental procedure, can get 100 colonies, 60% of them should be white spots, such as producing >20-40 blue spots, no colonies or few colonies, and there is a problem with the connection.
The results of the control experiment were good, but the target fragment was not recovered. What was wrong with the experiment?
A) The connection is incubated for 1 hour at room temperature to satisfy most clones. To improve efficiency, 4oC is required overnight.
B) The insert is contaminated, causing the 3'-T to be deleted, or inhibiting the junction, inhibiting transformation. To do this, the insert was mixed with the pGEM-T positive control and ligated. If the number of colonies in the control is reduced, the insert is purified or re-prepared. If a large amount of blue spots are produced, the insert is contaminated with nuclease, and the pGEM-T or pGEM-T Easy vector 3'-T is deleted.
C) The insert is not suitable for connection. Gel-purified inserts sometimes occur due to excessive UV exposure. UV over-irradiation produces pyrimidine dimers that are not conducive to ligation and the DNA must be repurified.
D) The amplified product of the thermostable DNA polymerase with repair function has no A at the end, and the latter is required for pGEM-T or pGEM-T Easy vector cloning. Add Taq DNA polymerase and nucleotides to add A to the end. For details, check the pGEM-T pGEM-T Easy carrier technical data (TM042).
E) Highly repetitive sequences may be unstable, causing deletions and rearrangements in amplification. If the insert is found to be highly frequent in deletion and rearrangement, recombinant defective E. coli strains such as SURE cells are required.
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