Below protocol is "A-beginners-guide-to-ChIP" from Abcam

3. Determination of DNA conc part (after "1.Cross-linking ~ 2.Sonication" )

Summarized protocol is :

1.11 : PCR purification

1.12 : RNase A + PCR purification

1.13 : proteinase K + PCR purification

1.14 : DNA conc determination

Question : I could not understand why PCR purification step is repeated 3times. And this protocol spent too much times.

Is there a reason to do this?

------Original protocol ---------

Determination of DNA concentration

1.11. The INPUT samples are used to calculate the DNA concentration for subsequent IPs. Purify the DNA using either a PCR purification kit or phenol:chloroform.

1.12. Add 2 μl RNase A (0.5 mg/ml). Heat with shaking at 65°C for 4-5 hr (or overnight) to reverse the cross-links. Purify the DNA

using a PCR purification kit according to the manufacturer’s instructions. The samples can be frozen and stored at -20°C.

Samples are treated with RNase A as high levels of RNA will interfere with DNA purification when using the PCR

purification kit. Yields can be severely reduced as the columns become saturated.

1.13. Add 5 μl proteinase K (20 mg/ml). Heat with shaking at 65°C for 4-5 hr (or overnight) to reverse the cross-links. Extract the

DNA with phenol:chloroform followed by ethanol precipitation in the presence of 10 μl glycogen (5 mg/ml). Resuspend in 100 μl

water. The samples can be frozen and stored at -20 °C.

Samples are treated with proteinase K, which cleaves peptide bonds adjacent to the carboxylic group of aliphatic

and aromatic amino acids. Cross-links between proteins and DNA are disrupted to aid DNA purification.

1.14. To determine the DNA concentration, transfer 5 μl of the purified DNA into a tube containing 995 μl TE to give a 200 fold

dilution and measure the OD260. Calculate the DNA concentration of the chromatin preparation in μg/ml.

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