Alkaline Lysis Mini Plasmid Preps

  1. Grow O/N in 1.5 ml LMM or Terrific broth (see Reagents) with 75µg/ml Amp
  2. Pour into ependorf tube and spin down cells at 7-8K for 2 min
  3. Aspirate s/n and resuspend in 50µl 25mM Tris pH 8, 10 mM EDTA; leave lids open
  4. 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
  5. Add 75µl KoAc solution and vortex
  6. Add 100µl of phenol/CHI3, close lids, vortex
  7. Spin 13K for 2 mins
  8. Remove supernatant, add to 500µl ethanol. Vortex and spin at 13 K for 5 min
  9. Aspirate s/n, removing all ethanol
  10. Resuspend in 50µl TE
  11. 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

DNA Isolation for Low-Melting Point Agarose (using elu-tip method)

  1. Excise fragment from gel and estimate volume.
  2. Add 1/100 volume of 1 M Tris pH 7.5, 1/50 volume of 0.5 M EDTA, and 1/100 volume of 5M NaCl.
  3. Incubate at 68°C for 10 minutes.
  4. Vortex and remove to a small tube.
  5. Incubate at 37°C for 5 minutes.
  6. Phenol extract 2-3 times (phenol at 42°C, spin at 13 K for 2 minutes.)
  7. Ether extract 1 time. Place tubes at 65°C, then speed-vac 2 to 5 minutes. Repeat procedure about 4 times to get rid of residual ether.
  8. Ethanol precipitate DNA and suspend pellet in 1 ml of low salt buffer (from elu-tip column protocol)
  9. Following the elu-tip protocol booklet, wash the column by pushing 5 ml of low salt buffer through the matrix at a rate of 0.5-1.0 ml/minute. The column may be incubated in the low salt buffer ³ 2 hours to improve recovery.
  10. Load DNA sample onto the column slowly (1-2 drops/second). NOTE: When recovering DNA from low-melt temperature agarose, use of the pre-filter is not recommended. Consult the protocols booklet for specific parameters of different types of nucleic acid purification (i.e. DNA purification when LMP agarose isn't used).
  11. Wash the column with 2-3 mls of pre-warmed (42°C) low salt buffer.
  12. Elude DNA with 0.4 ml of high salt buffer. Do a total of 2-3 washes as desired to be certain of good recovery.
  13. Ethanol precipitate DNA and resuspend in TE or dH2O.

Transformation Protocol Using Heat Shock

  1. Take competent E.coli cells from –80oC freezer.
a. Use DH5α cells in most cases.
b. If want to cut at XbaI or other DAM- enzyme site, use SCS110 cells which are deficient in Dam and Dcm methylases.
  1. Turn on water bath to 42οC.
  2. Put competent cells in a 1.5 ml tube (Eppendorf or similar). For transforming a DNA construct, use 50 ul of competent cells. For transforming a ligation, use 100ul of competent cells. You may need more or less cells, depending how competent they are.
  3. Keep tubes on ice.
  4. Add 50 ng of circular DNA into E.coli cells. Incubate on ice for 10 min. to thaw competent cells.
  5. Put tube(s) with DNA and E.coli into water bath at 42οC for 45 seconds.
  6. Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells.
  7. Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37 οC.(Can incubate tubes for 30 minutes, unless trying to grow DNA for ligation which)is more sensitive. For ligation, leave tubes for 1 hour.)
  8. Spread about 100 ul of the resulting culture on LB plates (with appropriate antibiotic added – usually Ampicillin or Kanamycin.) Grow overnight.
  9. Pick colonies about 12-16 hours later.

Double ligation (Richard Lab) Procedure

  1. Mix together the following:
a. 5μl digested front part (EcoRI and SpeI).
b. 5μl digested rear part (XbaI and PstI).
c. 4μl desionized water.
d. 3μl digested vector (EcoRI and PstI)
e. 2μl of Reaction Buffer for T4 Ligase.
f. 1μl of DNA Ligase.
  1. Incubate at room-temp for 10 mins (or at 4ºC overnight)
  2. Stop Reaction for 15 min at 80ºC