Create Plasmid_Prep_Without_Kit.md

Plasmid_Prep_Without_Kit.md

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+## Plasmid MidiPrep Without Use of Qiagen Kit
+
+For some of our low-copy plasmid strains, the Qiagen Midi kit has failed to retrieve any usable plasmid, and when one needs a significant amount of plasmid, a multitude of minipreps can be expensive and time-consuming. This method will allow for significant plasmid yield without contamination of genomic DNA, and importantly, it is simple and cost effective.
+
+### Reagents Required
+
+Solution 1 = 10 mM EDTA pH 8.0, 1-20 μg/mL RNAase A. Store at 4°C. (Prepare 300 mL)
+* To bring pH up, drop small amounts of 10M NaOH into solution while reading pH. Check with pH strips as well.
+
+Solution 2 = (0.1 M NaOH, 1% SDS). Store at room temperature. (Prepare 200 mL)
+* 2 grams total of SDS
+
+Solution 3 = 250g/L Potassium Acetate, 15% vol/vol Acetic Acid. Store at 4°C. (Prepare 100 mL)
+* Dissolve the 25 grams in 50 mL first and then bring the water volume up to 85. Then add the acetic acid.
+
+### Regular Plasmid Prep (Miniprep)
+
+1. Grow 2 mL bacteria (or 1.7 mL if growing in 2 mL tubes) with vigorous shaking or rolling in LB broth.
+2. Transfer the saturated bacterial culture to a 2 mL microcentrifuge tube if culture was not grown in a 2 mL tube. Spin at ≥10,000xg for 30 seconds. Discard supernatant.
+3. Resuspend the pellet in 100 μL Solution 1 by vortexing or pipeting.
+4. Add 200 μL Solution 2 and mix by gentle swirling (it is very important to be very gentle or you
+will get E. coli genomic DNA contamination in your plasmid prep).
+5. Add 75 μL cold (4°C) Solution 3 and mix by gentle inversion (it is very important to be very
+gentle or you will get E. coli genomic DNA contamination in your plasmid prep).
+6. Place tubes in a rack stored at -20°C and store at -20°C for 1 minute.
+7. Spin at max speed (≥10,000xg) for 5 minutes.
+8. Transfer up to 375 μL of the supernatant to a clean 1.5 mL tube (avoid taking any white
+precipitate).
+9. Add 225 μL 100% isopropanol and mix by vortexing. Spin at max speed (≥10,000xg) for 5
+minutes. Carefully discard the supernatant by pouring it out. Briefly spin down and remove any
+residual supernatant with a pipet.
+10. Add ≥ 1 mL 70% ethanol. Then pour away the ethanol.
+11. Spin at ~10,000xg for 5 seconds. Pipet away the residual ethanol.
+12. Leave on bench horizontally to dry for 2-5 minutes or speedvac for 1 minute.
+13. Resuspend the pellet in 50 μL (or 50-100 μL) in one of the following: 2mM Tris pH 8.5, sterile
+H2O, TE.
+
+Protocol from Rahul Patharkar
+
+### Large Plasmid Prep (Miniprep)
+
+1. Grow 100 mL bacteria with vigorous shaking or rolling in LB broth.
+2. Transfer 40 mL into the saturated bacterial culture to a 50 mL conical tube if culture was not grown in a 50 mL tube. Spin at ≥10,000xg for 5 minutes. Discard supernatant.
+3. Resuspend the pellet in 2000 μL Solution 1 by vortexing or pipeting.
+4. Add 4000 μL Solution 2 and mix by gentle swirling (it is very important to be very gentle or you
+will get E. coli genomic DNA contamination in your plasmid prep).
+5. Add 1500 μL cold (4°C) Solution 3 and mix by gentle inversion (it is very important to be very
+gentle or you will get E. coli genomic DNA contamination in your plasmid prep).
+6. Place tubes in a rack stored at -20°C and store at -20°C for 5 minutes.
+7. Spin at max speed (≥10,000xg) for 15 minutes.
+8. Transfer up to 375 μL of the supernatant to a clean 1.5 mL tube (avoid taking any white
+precipitate).
+9. Add 4 mL of 100% isopropanol and mix by vortexing. Spin at max speed (≥10,000xg) for 8
+minutes. Carefully discard the supernatant by pouring it out. Briefly spin down and remove any
+residual supernatant with a pipet.
+10. Add ≥ 4 mL 70% ethanol. Then pour away the ethanol.
+11. Spin at ~10,000xg for 5 seconds. Pipet away the residual ethanol.
+12. Leave on bench horizontally to dry for 2-5 minutes or speedvac for 1 minute.
+13. Resuspend the pellet in 300 μL in NF H2O.
+
+14. THe elution should yield 100 to 400 ng/uL.