problem
possible reason
Solution
No product or low yield
Template concentration is low or high
Electrophoresis detection template concentration, adjust template dosage
Template degradation
Re-preparation; genomic DNA, cDNA should be stored in small amounts and then cryopreserved
The template contains impurities that inhibit the reaction
Purification template
Insufficient primer concentration
Adjust primer concentration, especially for long fragment PCR
Primer has secondary structure
Redesign primers to avoid secondary structure; optimize annealing temperature
Primer degradation
The primers should be packed in high concentration and small amount, and stored at -20 °C to avoid repeated freezing and thawing.
Low Mg2+ concentration
Properly increase the concentration of Mg2+ from 1 mM to 3 mM at 0.5 mM intervals, and explore the optimal Mg2+ concentration for different templates and primers.
dNTP degradation
Store at -20 °C, small amount of packaging, avoid repeated freezing and thawing
Low enzyme purity and poor amplification efficiency
Use high quality DNA polymerase
Insufficient enzyme
Properly increase the amount of enzyme
Improper use of enzymes
Select the appropriate enzyme for the template and target fragment (see PCR Product Selection Guide for details)
Buffer failure
Replace buffer or use premixed PCR reaction system
Template denaturation is insufficient
Properly extend the denaturation time; increase the initial denaturation temperature to 98 °C for high GC or complex structural templates
High annealing temperature
Reduce the annealing temperature, which should be at least 5 °C lower than the primer Tm
Insufficient extension time
Increase the extension time, especially for long fragment PCR
Not enough number of cycles
Increase the number of cycles
PCR tube contamination
Reliable PCR tubes usually do not need to be sterilized, otherwise they should be autoclaved first. The used PCR tubes can not be washed and reused.
PCR instrument failure
Check program and module temperature
other
Use PCR enhancer
Too many non-specific products
System pollution
Design a control experiment to find the source of pollution, and pay attention to avoid cross-contamination during operation.
Primer concentration is too high
Properly reduce primer concentration
Primer sequence specificity
Redesign primer
Mg2+ concentration is too high
Decrease the Mg2+ concentration from 1 mM to 3 mM at 0.5 mM intervals and explore the optimal Mg2+ concentration for different templates and primers
dNTP concentration is too high
Reduce dNTP concentration
Excessive amount of enzyme
Reduce the amount of enzyme, decreasing at 0.5 U intervals
Annealing temperature is too low
Increase annealing temperature
Extension time is too short
Increase the extension time in increments of 1 min
Too many cycles
Reduce the number of cycles
The template structure is too complicated or the target fragment is too long
Select the appropriate enzyme (see PCR product selection guide for details); use PCR enhancer
other
HotStart PCR
TouchDown PCR
Nested PCR
Primer dimer
Complementary sequence at the 3' end of the primer
Redesign primer
Primer concentration is too high
Reduce primer concentration
Template concentration is too low
Increase template concentration
Inappropriate annealing temperature
Optimized annealing temperature
Too many cycles
Reduce the number of cycles
other
HotStart PCR
Severe tailing
Primer concentration is too high
Adjust primer concentration
Primer sequence specificity is poor or homologous sequences are present on the template
Redesign primer
Template degradation
Re-preparation template
Template concentration is too high
Reduce template concentration
dNTP concentration is too high
Reduce dNTP usage
Mg2+ concentration is too high
Decrease the Mg2+ concentration from 1 mM to 3 mM at 0.5 mM intervals and explore the optimal Mg2+ concentration for different templates and primers
Excessive amount of enzyme or poor quality
Reduce the amount of enzyme, decreasing at 0.5 U intervals; replace the enzyme with reliable quality
Denaturation temperature is too low
Increase the denaturation temperature in 0.5 ° C increments
Annealing temperature is too low
Increase annealing temperature
Extended time is too long
Shorten the extension time
Too many cycles
Reduce the number of cycles, decrement by 2 cycle intervals
System pollution
Design a control experiment to eliminate pollution sources
other
HotStart PCR
TouchDown PCR
Nested PCR
False positive
Homology between the target fragment and the non-specific amplified fragment
Design a negative control and redesign the primer after confirming the primer problem
System pollution
Design a negative control to determine if there is contamination and remove the source of contamination
A safe and secure Parcel Drop Box, these smart post boxes feature an anti-fishing design so that delivered parcels can only be retrieved from the holding compartment using your unique key. The wall mounted kind allows you install on the wall at an optimal posting level (5ft) for extra security and ease of delivery. Custom made are acceptable.
Wall Mounted Parcel Box,Floor Standing Parcel Box,Large Parcel Box,Small Parcel Box,Flexible Parcel Box
TAISHAN YUEXIN INDUSTRIAL GROUP LIMITED , https://www.tsyuexin.com