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Team:Mexico-UNAM-CINVESTAV/Protocols
From 2010.igem.org
(Difference between revisions)
| Line 7: | Line 7: | ||
#Grow O/N in 1.5 ml LMM or Terrific broth (see Reagents) with 75µg/ml Amp | #Grow O/N in 1.5 ml LMM or Terrific broth (see Reagents) with 75µg/ml Amp | ||
#Pour into ependorf tube and spin down cells at 7-8K for 2 min | #Pour into ependorf tube and spin down cells at 7-8K for 2 min | ||
| - | + | #Aspirate s/n and resuspend in 50µl 25mM Tris pH 8, 10 mM EDTA; leave lids open | |
| - | + | #Add 100µl of freshly prepared 1% SDS, 0.2M NaOH (5ml = 100ul 10M NaOH added to 4.4ml DDW then 500µl 10% SDS). Add it forcefully and you don't need to vortex | |
| - | + | #Add 75µl KoAc solution and vortex | |
| - | + | #Add 100µl of phenol/CHI3, close lids, vortex | |
| - | + | #Spin 13K for 2 mins | |
| - | + | #Remove supernatant, add to 500µl ethanol. Vortex and spin at 13 K for 5 min | |
| - | + | #Aspirate s/n, removing all ethanol | |
| - | + | #Resuspend in 50µl TE | |
| - | + | #Digest 2-5ul, adding 1µl preboiled 10mg/ml RNAse A (see Reagents for preparation). | |
| + | |||
KOAc solution: | KOAc solution: | ||
- 60 ml 5M potassium acetate | - 60 ml 5M potassium acetate | ||
- 11.5 ml glacial acetic acid | - 11.5 ml glacial acetic acid | ||
- 28.5 ml DDW | - 28.5 ml DDW | ||
Revision as of 18:39, 26 October 2010
Alkaline Lysis Mini Plasmid Preps
- Grow O/N in 1.5 ml LMM or Terrific broth (see Reagents) with 75µg/ml Amp
- Pour into ependorf tube and spin down cells at 7-8K for 2 min
- Aspirate s/n and resuspend in 50µl 25mM Tris pH 8, 10 mM EDTA; leave lids open
- Add 100µl of freshly prepared 1% SDS, 0.2M NaOH (5ml = 100ul 10M NaOH added to 4.4ml DDW then 500µl 10% SDS). Add it forcefully and you don't need to vortex
- Add 75µl KoAc solution and vortex
- Add 100µl of phenol/CHI3, close lids, vortex
- Spin 13K for 2 mins
- Remove supernatant, add to 500µl ethanol. Vortex and spin at 13 K for 5 min
- Aspirate s/n, removing all ethanol
- Resuspend in 50µl TE
- Digest 2-5ul, adding 1µl preboiled 10mg/ml RNAse A (see Reagents for preparation).
KOAc solution: - 60 ml 5M potassium acetate - 11.5 ml glacial acetic acid - 28.5 ml DDW
