Paul.xu: Created page with "NID miniprep plasmid isolation procedure Spin down 2 ml~5 ml of bacterial cultures.
Add 200 µl E-Buffer.
Resuspend cells by votexing Incubate cell suspension at 65..."

2017-06-22T02:52:06Z

Created page with "NID miniprep plasmid isolation procedure Spin down 2 ml~5 ml of bacterial cultures.<br/>Add 200 µl E-Buffer.<br/>Resuspend cells by votexing Incubate cell suspension at 65..."

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NID miniprep plasmid isolation procedure

Spin down 2 ml~5 ml of bacterial cultures.<br/>Add 200 µl E-Buffer.<br/>Resuspend cells by votexing

Incubate cell suspension at 65°C for 5 min.

Spin at maximum speed for 10 min or until a tight bacterial pellet was formed.

Remove pellet with a pippet tip.

Add 200 ul isopropanol, mix.

Spin at max speed for 10 min at RT.

Remove liquid by dumping.

Add 200 ul 70% Ethanol. Spin for 1 min

Discard ethanol.

Add 50 ul EB or TE

E buffer: 5% sucrose, 20–50 mM EDTA, 50 mM Tris pH 8, 0.75 M NH4Cl, 0.5% IGEPAL CA-630 (or Triton X-100), lysozyme 100 µg/ml, and RNase A 25 µg/ml.

Add 20–50 mM CaCl2 to the E-buffer reduces extraction of chromosomal DNA and large plasmids, but greatly facilitates formation of cellular debris during centrifugation. 

100 X enzyme stock: 10 mg/ml lysozyme, 2.5 mg/ml of RNase A, 50% glycerol, 50 mM Tris pH 8, store at −20°C.

Ref: [http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023457 http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023457]
[[Category:Protocols]]

NID Miniprep - Revision history

Paul.xu: Created page with "NID miniprep plasmid isolation procedure Spin down 2 ml~5 ml of bacterial cultures.Add 200 µl E-Buffer.Resuspend cells by votexing Incubate cell suspension at 65..."

Paul.xu: Created page with "NID miniprep plasmid isolation procedure Spin down 2 ml~5 ml of bacterial cultures.
Add 200 µl E-Buffer.
Resuspend cells by votexing Incubate cell suspension at 65..."