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Q&A
Can the Gel/PCR DNA Kit completely remove primers or dimer products from PCR reactions?
The Gel/PCR DNA Fragments Extraction Kit can only effectively remove (>90%) primers of less than 40 bp. When primers or dimer products are of more than 40 bp, they cannot be effectively removed. In this case, separate the PCR product from the dimer products by electrophoresis, excise the gel slice containing the desired product and purify it using Gel/PCR DNA Fragments Extraction Kit.
Can the Gel/PCR DNA Fragments Extraction Kit be used to clean up DIG-labeled DNA fragment?
Yes, DF Buffer does not affect the chemically linked DIG on dNTP. Similarly, this system can be used to clean up 32P-labeled DNA fragments.
A band, smaller than the desired DNA fragment is present in the eluant.
The smaller band may be a single-stranded form of the PCR product. The occurrence of it could be due to the elongation of the PCR product not being complete or the PCR product is denatured during the preparation. In this case, to re-anneal the single-stranded DNA, incubate the solution at 95ºC for 2 minutes and let it cool slowly to room temperature. The re-annealed PCR product can be used as usual in all downstream applications.
Why DNA recovery is lower than expected?
1) Gel slice did not dissolve completely: Gel slice was too big. If using more than 300 mg of gel slice, separate it into multiple tubes. Raise temperature of incubation to 60°C and extend incubation time.
2) Incorrect DNA Elution Step: Ensure that Elution Buffer was added and absorbed to the center of the DF Column matrix
3) Incomplete DNA Elution: If the sizes of DNA fragments are larger than 10 Kb, use preheated Elution Buffer (60-70°C) during the Elution Step to improve the elution efficiency.
4) Do not overload the column with too much DNA. Higher recovery is attained when lower amount of DNA is loaded. Divide the large amount of DNA into more than one column.
5) If ddH2O is used for elution, make sure that its pH is between 7.0 and 8.5, as pH lower than 7 leads to lower elution efficiency.
6) Make sure that complete DNA elution takes place by adding no less than 30 ml of elution solution onto the membrane and letting it completely absorb into the membrane before centrifugation.
7) Large DNA fragments are eluted less readily than small DNA fragments. When the DNA product is larger than 5 Kb, use the elution solution preheated to 60ºC.
Why doesn't the Eluted DNA perform well in downstream applications?
1) Residual ethanol contamination: After the wash step, dry the DF Columnn with additional centrifugation at top speed for 5 minutes or incubate at 60°C for 5 minutes.
2) DNA was denatured (a smaller band appeared on gel analysis): Incubate eluted DNA at 95°C for 2 minutes, and then cool slowly to re-anneal denatured DNA.