PureLink™ HiPure Plasmid Midiprep Kit

PCR条件の最適化について、いくつかの例を以下に記します:

-プライマーの3’末端付近で相補的な配列がないか確認する。
-アニーリング温度を2-5℃ほど上げ、アニーリング時間も短くする。最初の数サイクルでアニーリング温度に設定し、それ以降は低いアニーリング温度でサイクルを回す。
-Try hot-start protocols. 　　ホットスタートプロトコルを試す。
-それぞれのプライマーや鋳型の組み合わせに応じてマグネシウム濃度を最適化する。
-汚染DNAからの増幅が起こらないようにするために、エアロゾル混入防止のフィルターつきチップや 　　UDG（ウラシルDNAグリコシラーゼ）を使用する。

A common problem encountered with absorbance measurements is turbidity of samples. (This could be caused by residual resin from the column.) If there is insoluble material in the cuvette (not often detected by the naked eye), much of the UV light is not absorbed but scattered, leading to an artificially high UV absorbance reading (at 260 or 280 nm, for example.) If your A260 is high, we recommend that you check the A320 to determine if there is resin in the sample. You can also try to centrifuge or filter (0.2 µm filter) your sample to remove any resin and then recheck the concentration.

Yes, we would recommend purchasing the PureLink HiPure BAC Buffer Kit (Cat. No. K210018). This kit includes Resuspension Buffer (R3) (250 ml), Lysis Buffer (L7) (250 ml), Precipitation Buffer (N3) (250 ml), and RNase A (20 µg/ml) (5 ml).
You will need to add less RNase A than stated on the bottle label of the R3 buffer in this kit. It says to add 5.6 mL of RNase A. This is the correct amount for the BAC protocol; however, if you are performing standard plasmid isolation, 1.4 mL RNase A should be added.

The HiPure kits should remove all protein from the DNA including endonucleases. For the silica-based PureLink Quick Plasmid Miniprep Kit, we recommend an extra wash with the optional Wash Buffer W10 to remove endonucleases. This solution is not compatible with the HiPure system and should not be used with those kits. Alternatively, heat the eluted DNA in TE for 10 min at 70 degrees C. This should heat-inactivate any contaminating nucleases.

Extra bands can occur when plasmid DNA is nicked and/or permanently denatured. Plasmid DNA that has been nicked (covalently opened) will run slower than supercoiled DNA during electrophoresis. A small amount of this species of DNA is common and is suitable for downstream applications. Permanently denatured DNA will migrate ahead of the supercoiled DNA and may not be suitable for downstream applications. Do not allow the lysis reaction to proceed longer than 5 minutes.