https://eln.excellgen.com/index.php?action=history&feed=atom&title=Plasmid_MiniPrep_NIDPlasmid MiniPrep NID - Revision history2026-10-19T07:24:27ZRevision history for this page on the wikiMediaWiki 1.44.0https://eln.excellgen.com/index.php?title=Plasmid_MiniPrep_NID&diff=7793&oldid=prevPaul.xu at 17:48, 9 January 20132013-01-09T17:48:08Z<p></p>
<p><b>New page</b></p><div>NID miniprep plasmid isolation procedure <br />
<br />
1. Spin down 2 ~ 5 ml of&nbsp; bacterial cultures.<br> <br />
<br />
2. Add 200 ul extraction buffer, transfer to 1.5 ml microcentrifuge tube. Resuspend into homogenous solution.<br> <br />
<br />
3. Incubate at 65°C for 5 min.<br> <br />
<br />
4. Spin at 13000 rpm for 5 ~ 10 min.<br> <br />
<br />
5. Remove pellet using pippet tip.<br> <br />
<br />
6. Add 150 µl isopropanol, mix.<br> <br />
<br />
7. Spin at 13000 rpm for 10 min at RT.<br> <br />
<br />
8. Discard liquid.<br> <br />
<br />
DNA usually forms film-like precipitates that adhere well to tube walls and are invisible in isopropanol solutions<br> <br />
<br />
9. Add 100 70% ethanol. Gently remove ethanol.<br> <br />
<br />
10. Dissolve DNA pellet in 50 µl TE buffer. <br />
<br />
<br> <br />
<br />
DNA can be used for restriction digestion. For sequencing: DNA may have to be purified using silica spin column.'''<br>''' <br />
<br />
<br> '''Extraction Buffer'''<br> <br />
<br />
5% sucrose<br> <br />
<br />
50 mM EDTA<br> <br />
<br />
50 mM Tris pH 8<br> <br />
<br />
0.75 M NH<sub>4</sub>Cl or Ammonium acetat (NH4Ac)<br> <br />
<br />
20 mM CaCl<sub>2</sub><br> <br />
<br />
0.5% IGEPAL CA-630 (Nonidet-P40) or Triton X-100<br> <br />
<br />
Add lysozyme 100 µg/ml, and RNase A 25 µg/ml from 100X enzyme stock (10 mg/ml lysozyme and 2.5 mg/ml of RNase A&nbsp; in 50% glycerol, 50 mM Tris pH 8).<br> <br />
<br />
2013/1/9<br />
<br />
digest with Nde1+Bgl11<br />
<br />
[[Image:V19_digest_1-9-2013.JPG]]<br />
<br />
[[Category:Protocols]]</div>Paul.xu