Editing Plasmid MaxPrep DNA using Diatomaceous Earth (section)

== MaxiPrep  ==

1. Inoculate 1L mL 2xYT or LB/AMP or Kan from 1-5 mL overnight<br>2. Grow at 37 oc overnight. <br>3. Spin cells at 6000 rpm for 10 min at 4ºC using GS3 rotor.<br>4. Resuspend cells in 20 mL P1 without RNase A.<br>5. Add 20 mL P2. Stir very gently using a long 5 ml plastic pippette, until homogenous and clear.<br>6. Add 20 ml P3, mix, add 100 mg/ml RNAse sol to 0.1 mg/ml. Leave it on bench to digest o/n<br>7. Spin 6000 rpm at 4ºC for 10 min.<br>8. Filter supernatant using a coffee filter paper.<br>10. Add 4 M CaCl2 to final conc of 0.5 M, incubate at RT for 10 min. Spin at 25 oC (RT) for 10 min at 5000 rpm.<br>11. Add 0.6 volume isopropanol or 2 volume of ethanol. Invert to mix. Leave it at room-temperature for 10 min.<br>12. Spin at 6000 rpm at 4ºC or 25 ºC for 10 min. <br>13. Resuspend pellet in 5X1 ml PB,&nbsp; combine liquid and transfer to a clean 50 ml tube. <br>(14. Add 10% Triton X-110 to final concentration of 0.1% TritonX-114 to the plasmid DNA solution, mix by votexing.)<br><br>15. Add 5 ml stock SiO2&nbsp; slurry. Incubate at RT for 1 hour.<br>20. Spin down SiO2 particles at 5000 rpm for 5 min.<br>21. Wash SiO2 particles with 2 times PB buffer than 2 times with 15 ml of '''Washing Solution (PE). Spin''' at 5000 rpm for 5 min.<br> 

PE: 20 mM Tris-Cl pH 7.5, 2 mM EDTA, 0.4 M NaCl, and 50% v/v ethanol<br> 

<br>22. Wash SiO2 particles with 5 ml of 80% ethanol. Spin at 5000 rpm for 5 min and remove liquid<br>23. Add 2 ml of water or TE preheated to 65-70 oC, and wait 2 min (or place on hot bath for 2 min)<br>24. Spin at 5000 rpm for 5 min.<br>25. Transfer liquid containing DNA to a 1.5 ml tube for storage.<br>26. Measure DNA concentration and run 5 ul on agarose gel to check for the presence of RNA. 

<br>'''P3 (~5.5) [500 mL]'''<br>147g KOAc (3M)<br>55 mL glacial HOAc<br><br> 

Precipitate RNA using CaCl2. Add 43 ul, 100 ul and 120 ul of 4 M CaCl2 to 300 ul filter solution from step 8, incubate at RT for 10 min, spin using microcentrifuge for 10 min.<br>

Transfer supernatant to another tube. Add 300 ul isopropanol. incubate at RT for 15 min on bench, spin down pellet. Dissolve both Ca ppt and isopropanol ppt in 50 ul TE. Use 8 ul to run gel.

[[Image:Ca_ppt_RNA-7_4-14.JPG]]<br>

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Without Ca ppt

isopropanol ppt<br>

| supenatant of 0.5 M CaCl2, ppt with isopropanol <br>
| pellet of 0.5 M CaCl2 precipitation<br>
| supenatant of 1.0 M CaCl2, ppt with isopropanol <br>
| pellet of 1.0 M CaCl2 precipitation<br>
| supenatant of 1.5 M CaCl2, ppt with isopropanol <br>
| pellet of 1.5 M CaCl2 precipitation<br>
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