PCR Taq

• What are the functions of Taq DNA polymerase?
  1. PCR
  2. 3'A – tailing of blunt ends
  3. primer extension
  4. DNA labeling reactions
• What characteristics does GenScript Taq DNA polymerase have?
  1. High Stability
  2. High PCR yield
• What is the recommended enzyme amount when using Taq DNA Polymerase or Green Taq DNA Polymerase?

  0.5 U or more

• How is Taq different from Green Taq?

  The Green Taq contains normal Taq with special stabilizers. The Green Taq can be stable under RT for up to two weeks.

• Can Taq DNA Polymerase or Green Taq be used to amplify GC-rich amplicons?

  No.

• Can Taq DNA Polymerase be used in other buffers?

  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).

• Which buffer should I use if I want to control the level of magnesium (Mg2+) in the reaction? Does the presence of Mg2+ inhibit PCR?

  This is a potential concern. For this reason we suggest to use Mg-free Taq DNA Polymerase (Cat. No. E00008).

• How should I set up an amplification reaction using Taq DNA Polymerase?

  Here are the guidelines for a 50 μl PCR reaction:

  1. 0.5 μl Taq DNA Polymerase
  2. 1 μl 10 mM dNTP
  3. 1 μl each primer
  4. 2 μl genomic template (up to 100 ng/μl)
  5. 5 μl 10x Taq Buffer (Standard Taq containing Mg2+ )
  6. 39.5 μl sterile or filtered H2O
  7. Denature at 94°C
  8. Extend 1 minute/kb
• What is the proper concentration for a routine PCR reaction?
  1. For up to 3 kb, we recommend 1.25 units per 50 µl reaction
  2. For specialized applications, including 3–8 kb amplicons, 2.5 units per 50 µl reaction is recommended
• What is the maximum product length that can be made by GenScript's Taq DNA Polymerase?

  Up to 8 kb λdna.

• What type of DNA end results from a primer extension reaction or a PCR using Taq DNA Polymerase?

  The Poly A tail contains sticky ends and can only be used in TA cloning.

• Why is there no product when visualized on an agarose gel?

  Potential reasons are improper set up of PCR system/ program, or problems with electrophoresis.

• The product sequence doesn't completely match the expected sequence. How can this result be improved?

  Try using a high-fidelity DNA Polymerase.

• When should Taq DNA Polymerase be used in a primer extension reaction or for PCR?

  The Taq can be used when the PCR reaction is started or after degeneration.

• Will the 5'→3' flap endonuclease activity of Taq DNA Polymerase degrade primers?

  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).

• Where can I find help troubleshooting my PCR?

  Please send all questions to product@genscript.com.

  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	Test the template with an established PCR system Repeat template purification to remove the remainder alcohol, Phenol or other contamination
  	Cycle conditions	Change annealing temperature Increase cycle number Prolong the elongation time
  	Primer problem	Both primers must have the same concentration. The final concentration must be between 0.2 µM ~ 0.6 µM Make sure that the primers are not degraded
  	Low enzyme concentration	Increase the amount of polymerase in 0.5 U steps
  	Low MgCl2 concentration	Increase the MgCl2 concentration in 0.25mM steps
  	Inadequate annealing temperature	Increase annealing temperature
  	Improper template concentration	Test serial dilution of template
  	Formation of primer dimers	When the temperature of the PCR cycle reaches 94°C insert PCR reaction tube and continue the program

PCR Cloning Kit

• What are the differences between the GenBuilder™ kit and the PCR Cloning Kit?

  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.

• Technical information is not available in the manual, why is that?

  Currently, we are waiting for a patent on GenBuilder. Once the patent is valid a more detailed explanation in the manual will be provided.

• Why don't I generate sufficient colonies when the GenBuilder™ reaction is transformed?

  This may be caused by four factors.

  1. The competent cells have low transformation efficiency.
  2. Too much reaction mixture is used.
  3. Presence of Inhibitory contaminants from PCR DNA or linearized vector.
  4. The molar ratio of vector to insert is off.
     Please refer to the manual for TROUBLESHOOTING.
• Why do I generate incorrect colonies when the GenBuilder™ reaction is transformed?

  Two factors may be involved.

  1. The cloning vector is not completely linearized.
  2. The cloning reaction is contaminated with plasmids having the same antibiotic resistance.
• What factors need to be considered when designing the primer?

  Two sets of primers are used to amplify the gene of interest:

  1. 15-bp homology regions to the vector, flanking the cloning site into the insert.
  2. Specific gene sequence. If the sequence has 5' overhang, the start of the 15 bp homology must begin from the 5' most extension to include the overhang; if the sequence has a 3' overhang, homology should begin where the DNA becomes double-stranded. Please refer to the manual for illustration of primer design.
• What should the purity of my primer be to be compatible with the GenBuilder™ cloning method?

  Desalted oligos (from a qualified supplier) are suitable for cloning with the GenBuilder™ PCR cloning Kit.

• Can multiple fragments be cloned into a single vector using this 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.

• Can I use GenBuilder™ PCR Cloning Kit with my own vectors?

  Yes. GenScript's GenBuilder™ PCR Cloning Kit has been successfuly tested with several commercial and non-commercial vectors.

• What factors need to be considered when using GenBuilder™ PCR Cloning Kit with customers' vector?

  We recommend linearizing your vectors before using GenBuilder™ PCR Cloning Kit. Once linearized, the columned and gel-purified vector is ready for GenBuilder™ reaction.

• Will the GenBuilder™ reaction work more efficiently if I use primers that contain a longer than 15 base region of homology?

  A 15-20 bp homology is recommended.

• What is the stability of the enzyme included in GenBuilder™ PCR Cloning Kit?

  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.

Plasmid Purification

• Why is my plasmid DNA yield low?
  1. Check if the bacteria was cultured properly.
  2. Check if bacteria cells were resuspended completely.
  3. Incubate the Elution Buffer at 30-60°C，this should help increase yields.
• Can the QuickClean II plasmid Miniprep Kit be used for isolating plasmid DNA from mammalian cells?

  No. The Kit provides a fast, simple, and cost-effective plasmid miniprep method for 1–5 ml of overnight cultures of E. Coli.

• What is the recommended culture medium for the Plasmid miniprep kit?

  LB culture medium

• How do I know if my plasmid is a high- or low copy number type?

  For example, a series of PUC vector is high-copy-number plasmid. You can know the different type of plasmid from their name.

• How to resolve genomic DNA contamination in my plasmid prep?

  If getting genomic DNA contamination in the prep, invert the tube gently after adding the Lysis Buffer.

• Can I use QuickClean Miniprep kits for low-copy plasmids and cosmids?

  The kit can purify the plasmid from low-copy plasmids, but from cosmids.

• Are GenScript buffers with identical names in different kits the same?

  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.

• Will QuickClean II PCR Purification Kit remove fluorescent dyes from real-time PCR reactions?

  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.

• Is it necessary to repeat the wash procedure?

  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.

• Can I use TE buffer or water to elute DNA from the column?

  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.

• What additional consumables does the user need?

  Ethanol is needed to prepare the wash solution for all three kits. Isopropanol is also needed for QuickClean II PCR or Gel Extraction Kit.

• Can I extract and purify DNA from gels using TAE running buffer?

  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.

• Can I extract and purify DNA from low melting point (LMP) Agarose gels?

  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	Please check if the bacteria was cultured properly. Please check if the bacteria cells were resuspended completely. 3. Incubate the Elution Buffer at 30～60°C，to help increase the yields.
  Absorbance problem	Absorbance is the difference of the sample from the blank, please use the Elution Buffer to adjust the blank to a zero value and use it to dilute the sample. If the ratio of OD260/ OD230 is too low, wash the spin column for one more time. In the case of a low ratio of OD260-320/ OD280-320, there may be protein contamination. In this case please add Neutralization Buffer, and then centrifuge buffer with sufficient rotating speed, thus to make precipitation compact; be careful to pipette supernatant and avoid pipetting the precipitate. If the ratio of OD260-320/ OD280-320 is too high, add more RNase A to Resuspension Buffer to a final concentration 100 μg/ml.
  Electrophoresis problem	If there is genomic DNA in the result, invert the tube gently (step 3 and 4). If there is RNA in the result, add more RNase A to Resuspension Buffer to a final concentration 100 μg/ml.