Can Gel/PCR DNA Fragments Extraction Kit be used to clean up
DIG-labeld DNA fragment?

Gel/PCR DNA Fragments Extraction Kit can only effectively (>90%) removes primers of less than 40-bp. When primers or dimer products are 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.

Yes, DF Buffer does not affect the chemically linked DIG on dNTP. Similarly,this system can be used to clean up 32 P-labeled DNA fragment.

The smaller band may be a single-stranded form of the PCR product. The occurrence of it could be due to that elongation of the PCR product is not completed or that PCR product is denatured during the preparation. In this case, to re-anneal the single-stranded DNA by incubating the solution at 95˚C for 2 minutes and let it cool to room temperature slowly. The re-annealed PCR product can be used as usual in all downstream applications

(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 DF Column matrix

(3) Incomplete DNA Elution

•  If the size of DNA fragments is larger than 10 kb, use preheated Elution Buffer (60 -70 ℃ ) on 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. Split the loading of high amount of DNA into more than one column.

(5) If ddH 2 O is used for elution, make sure than its pH is between 7.0 and 8.5. 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 adequately absorbed into the membrane before centrifugation.

(7) Large DNA fragment is eluted less readily than small DNA fragment. When the DNA product is larger than 5-kb, please preheat elution solution to 60˚C before Elution step.

(1) Residual ethanol contamination

•  After washing step, dry DF Column with additional centrifugation at full speed for 5 minutes or incubation at 60 ℃ for 5 minutes.

(2) DNA was denatured (a smaller band appeared on gel analysis)

•  Incubate eluted DNA at 95 ℃ for 2 minutes, than cool down slowly to re-anneal denatured DNA.