What is the starting quantity of DNA material to be PCR?
Recommendations of Template DNA in a 50 ul reaction volume
| DNA Type | Amount of DNA |
| Human genomic DNA | 0.1 to 1 ug |
| Plasmid DNA | 0.5 to 5 ng |
| Phage DNA | 0.1 to 10 ng |
| E.coli genomic DNA | 10 to 100 ng |
What is the shelf life of this item?
Minimum Shelf Life: 1 year
Can exTEN handle GC rich template? What's percentage limit?
Yes, exTEN has been able to produce high GC fragments up to 75% with the addition of PCR enhancers such as DMSO and Betaine.
Does multiple freeze thaw cycle affects the activity of polymerase?
Yes. It is recommended to distribute the master mix to smaller volumes.
What is the error rate for this PCR mastermix?
It is slightly lower than Taq Polymerase.
Advantages of this PCR master mix?
This premix formulation saves time and reduces contamination by reducing the number of pipetting steps for PCR setup. Higher yield and able to amplify longer PCR products.
What is the speed/extension rate for exTEN?
1 minute per kb.
Does exTEN possesses proof-reading function?
exTEN consists of a small percentage of a polymerase which exhibits 3’ 5’ exonuclease (proofreading) ability.
Can exTEN be used for colony PCR?
Yes.
Will the green loading dye inhibit PCR reaction?
No. The green loading dye does not pose any inhibitory effects during PCR.
After PCR, can we directly load the products into gel since loading dye is included in this master mix?
Yes. exTEN consists of a density reagents and 2 tracking dyes which migrate at the same rate as a 4000bp and 100bp DNA fragment in a 1% agarose gel.
Can we remove the loading dye from the amplified product for downstream processes such as Sanger sequencing?
Yes. Commercial PCR cleanup kits can be used.
Are the amplified fragments sticky ended or blunt ended?
A majority of the PCR products generated contain a 3’A overhang while a small percentage are blunt-ended.
Can the dye be removed using column based clean up kit?
Yes. Commercial PCR cleanup kits can be used.
Can I reduce the recommended reaction volume to half?
It is recommended to perform PCR reaction setups at 50µl volumes.
Can exTEN be used for multiplex PCR?
No.
Is exTEN a hifi DNA polymerase?
Not exactly. The amplification effciency of exTEN is enhanced through a lower error rate of misincorporated nucleotides compared to just Taq DNA Polymerase alone.