- PCR
- 3'A – tailing of blunt ends
- primer extension
- DNA labeling reactions
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0.5 U or more
The Green Taq contains normal Taq with special stabilizers. The Green Taq can be stable under RT for up to two weeks.
No.
Taq DNA polymerase can have 100% activity in the supplied GenScript Taq Buffer. Additionally, the Taq DNA polymerase can also work well in other buffers. For your convenience, GenScript offers Taq buffer under the name 10x Taq Buffer (Cat. No. B0005).
This is a potential concern. For this reason we suggest to use Mg-free Taq DNA Polymerase (Cat. No. E00008).
Here are the guidelines for a 50 μl PCR reaction:
Up to 8 kb λdna.
The Poly A tail contains sticky ends and can only be used in TA cloning.
Potential reasons are improper set up of PCR system/ program, or problems with electrophoresis.
Try using a high-fidelity DNA Polymerase.
The Taq can be used when the PCR reaction is started or after degeneration.
No. The exonuclease will only degrade double stranded DNA that it encounters while extending a DNA fragment. It will degrade a secondary primer if bound to the same strand (e.g. a mutagenesis primer).
Please send all questions to [email protected].
Problem
|
Possible Cause(s)
|
Solution(s)
|
---|---|---|
Little or no amplification product
|
GC rich template
|
add 5% DMSO, 1 M betaine, or both can be included in PCR reactions
to
improve
results
|
Template degradation
|
Analyze the template on an agarose gel to check for possible degradation |
|
PCR inhibitor contamination
|
|
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Cycle conditions
|
|
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Primer problem
|
|
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Low enzyme concentration
|
Increase the amount of polymerase in 0.5 U steps
|
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Low MgCl2 concentration
|
Increase the MgCl2 concentration in 0.25mM steps
|
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Inadequate annealing temperature
|
Increase annealing temperature
|
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Improper template concentration
|
Test serial dilution of template
|
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Formation of primer dimers
|
When the temperature of the PCR cycle reaches 94°C insert PCR
reaction tube
and
continue the program
|
GenBuilder™ seamless cloning technology offers high efficiency, quick and direct cloning, bypassing all the tedious procedures of traditional cloning that often involve restriction, ligation, and sometimes phosphorylation. This technology works with any DNA sequence and any vector. The only requirement is that the PCR primers have a sequence containing 15 or more nucleotides homologous to the vector sequence.
Currently, we are waiting for a patent on GenBuilder. Once the patent is valid a more detailed explanation in the manual will be provided.
This may be caused by four factors.
Two factors may be involved.
Two sets of primers are used to amplify the gene of interest:
Desalted oligos (from a qualified supplier) are suitable for cloning with the GenBuilder™ PCR cloning Kit.
Yes. GenBuilder™ PCR Cloning Kit can be used for multiple fragment recombination if there are not many repeated sequences among multiple fragments. However, we recommend recombination one fragment at a time. Several performances of recombining 2 - 3 fragments demonstrated efficiency as low as 20%-30%, and lead to a direct repeat deletion.
Yes. GenScript's GenBuilder™ PCR Cloning Kit has been successfuly tested with several commercial and non-commercial vectors.
We recommend linearizing your vectors before using GenBuilder™ PCR Cloning Kit. Once linearized, the columned and gel-purified vector is ready for GenBuilder™ reaction.
A 15-20 bp homology is recommended.
The liquid enzyme should be stored at -20°C for at least 12 months without activity loss.
Problem
|
Probable Cause
|
Solution
|
---|---|---|
Few or no colonies are obtained from the transformation.
|
The competent cells have low transformation efficiency.
|
Check the transformation efficiency. Competent cells with >1×108
cfu/μg
are
recommended.
|
Too much reaction mixture is used.
|
Do not add more than 20 μl of reaction mixture to 50 μl of
competent cells.
Too
much reaction mixture inhibits the transformation.
|
|
There are inhibitory contaminants from PCR DNA or from linearized
vector.
The
molar ratio of vector to insert is off.
|
Both the PCR DNA and the linearized vector should be purified.
Usually an
insert/vector molar ratio of 2:1 is optimal. If the insert is as
large as
the linearized vector,
a
molar ratio of 1:1 can also be used.
|
|
Too long or short a recombination time.
|
It is recommended to keep the recombination procedure within 30
min.
|
|
The linearized cloning vector or primer is large.
|
Connect the product to the target step recombinant vector.
|
|
The cloning vector is not completely linearized.
|
Gel-purify the linearized vector.
|
|
The cloning reaction is contaminated with plasmids having the same
antibiotic
resistance.
|
Purified PCR DNA may contain the template plasmid, so gel-purify
the PCR
DNA.
|
|
Most of the colonies contain no insert.
|
The cloning vector is not completely linearized.
|
Gel-purify the linearized vector.
|
The cloning reaction is contaminated with plasmids having the same
antibiotic
resistance.
|
Purified PCR DNA may contain the template plasmid, so gel-purify
the PCR
DNA.
|
No. The Kit provides a fast, simple, and cost-effective plasmid miniprep method for 1–5 ml of overnight cultures of E. Coli.
LB culture medium
For example, a series of PUC vector is high-copy-number plasmid. You can know the different type of plasmid from their name.
If getting genomic DNA contamination in the prep, invert the tube gently after adding the Lysis Buffer.
The kit can purify the plasmid from low-copy plasmids, but from cosmids.
Yes, GenScript buffers that have exactly the same name, are chemically identical and can be exchanged between kits. For example, wash solution and elution buffer from the QuickClean II PCR Purification Kit are exactly the same as wash solution and elution buffer from QuickClean II PCR Gel Extraction Kit.
Yes, QuickClean II PCR Purification Kits remove SYBR Green dye efficiently from the real-time PCR reaction. The kit will also remove fluorescent dye labeled dNTP used for PCR DNA labeling, such as Cy3-dUTP, etc.
The DNA eluted from the column is pure enough for most downstream applications. However, if the downstream applications are sensitive to salt carryover, it is better to wash the column twice prior to elution.
Elution Buffer is 2.5 mM Tris-HCl pH 8.5. TE buffer or water can also be used, but yield will be slightly lower. The pH of the elution solution is critical; buffers with a higher pH such as 8.5 or above will be more efficient to elute DNA from the column.
Ethanol is needed to prepare the wash solution for all three kits. Isopropanol is also needed for QuickClean II PCR or Gel Extraction Kit.
Yes. QuickClean II PCR or Gel Extraction Kit can be used to extract and purify DNA from gels using either TBE or TAE as the electrophoresis buffer.
Yes. QuickClean II PCR or Gel Extraction Kit can be used to extract and purify DNA from low melting point (LMP) gels.
Problem
|
Solution
|
---|---|
Obtain no plasmid DNA
|
If there is no plasmid DNA in the elution buffer, please check whether
the ethanol
had
been added to wash buffer according to the volume marked on bottle
label.
|
Low plasmid DNA yields
|
|
Absorbance problem
|
|
Electrophoresis problem
|
|