X-ray Crystallography Core Laboratory

Phenol/chloroform extraction with ethanol precipitation (removes proteins and other contaminants from DNA)
 

  1. Add 50 ul chloroform, 50 ul buffer-saturated phenol, and TE buffer to a total volume of 300 ul.
  2. Vortex and spin at 14K for 5 min.
  3. Remove 180 ul of the supernatant to a fresh microfuge tube.
  4. Add 1/10 volume (18 ul) of Na acetate pH 5.2.
  5. Add 2.5 volumes (450 ul) of 100% ethanol (-20°C)
  6. Vortex and freeze for 30 min.
  7. Spin 30 min at 14K.
  8. Pour off supernatant and wash with 1 ml 70% ethanol (-20°C).
  9. Spin 5 min at 14K.
  10. Pour off supernatant and spin in the Speed-Vac or vacuum dry for 10 min.
  11. Add TE buffer to 20 ul.


Competent cells
 

  1. Grow a 5 ml overnight culture.
  2. Dilute to 50 ml with LB.
  3. Grow for 1 hour at 37°C with shaking.
  4. Pellet at 3K for 5 min.
  5. Resuspend in 25 ml ice-cold 0.1M CaCl2.
  6. Pellet at 3K for 5 min to wash.
  7. Resuspend in 2.5 ml ice-cold 0.1M CaCl2.
  8. Leave on ice for 1 hour.
 
Transformation
 
  1. Add 5 ul of DNA to 200 ul competent cells.
  2. Incubate on ice for 1 hour.
  3. Heat shock at 42°C for 1 - 3 min.
  4. Incubate on ice for 3 min.
  5. Add 1 ml of LB and incubate at 37°C for 1 hour.
  6. Spin cells at 3000 RPM for 5 minutes. Pour off the supernatant, then resuspend cells in the remaining LB.
  7. Plate the entire volume of resuspended cells on LB/(your antibiotic) plate.
 
Novagen protocol
 
  1. Add 1 ul of DNA to 20 ul competent cells.  Stir gently.
  2. Incubate on ice for 5 min.
  3. Heat shock at 42°C for 30 sec.
  4. Incubate on ice for 2 min.
  5. Add 80 ul room temperature SOC medium.
  6. Incubate at 37°C for 30 min.
  7. Plate 50 ul on LB/(your antibiotic) plate.
 
LB/Amp plates
 
  10g/L bactotryptone
  5g/L yeast extract
  10g/L NaCl
  16g/L bactoagar
 
Autoclave and when cooled add ampicillin to 100 ug/ml.
 

Restriction digest
 
  10 ul vector (1 ug/ul)
  3 ul 10X reaction buffer
  0.5 ul 100X BSA
  0.1 ul RNAse
  1 ul enzyme 1
  1 ul enzyme 2
  14.4 ul water
  30 ul total volume
 
Digest for 2 hours at 37°C.
Add 0.5 - 1 ul CIP (calf intestinal phosphatase) to the vector.  Incubate 30 min at 37°C.
 
  5 ul insert
  3 ul 10X reaction buffer
  0.5 ul 100X BSA
  0.1 ul RNAse
  1 ul enzyme 1
  1 ul enzyme 2
  19.4 ul water
  30 ul total volume
 
Digest for 2 hours at 37°C.
 

Ligation
 
1 ul vector
2 ul insert
2 ul 10X ligation buffer
1 ul ligase
14 ul water
20 ul total volume
 
Incubate at 12°C in the heating block in the cold room overnight.
 

PCR
 
  1 ul template DNA (~100 ng/ul)
  5 ul 10X PCR buffer
  3 ul forward oligo (100 ug/ul)
  3 ul reverse oligo (100 ug/ul)
  5 ul dNTP's (1.5 mM)
  1 ul Pfu polymerase
  32 ul water
  50 ul total
 
Typical scheme:
 
  25 cycles - 1 min at 94°C, 1min at 60°C, 2 min per kb at 72°C
 
* begin restriction digest before purifying on a gel, if needed
 

Agarose gel electrophoresis
 

  1. Prepare 0.5 L of TAE buffer from the 50X stock.
  2. Microwave 0.75g of agarose in 50 ml of TAE buffer (1.5% gel) for 45 sec.
  3. After cooling gel to lukewarm add 2 ul of 10 mg/ml ethidium bromide.
  4. Place comb in gel mold and add the gel.
  5. Cool gel until it is solid and add the TAE buffer (with 20 ul of 10 mg/ml ethidium bromide) to the well.
  6. Load samples and run at 95V until the running dye reaches the end of the gel.

Purifying DNA from agarose gels
 
  1. Cut out bands of interest from the agarose gel and freeze the gel slice for 1 hour at -20°C.
  2. Thaw at 37°C for 5 min.
  3. Extrude the gel through a 1 ml syringe into the sample cup of an Ultrafree-MC filter unit to thoroughly macerate the gel.
  4. Centrifuge at 8K for 20 min.
  5. Ethanol precipitate the eluted DNA.
 
 
  Plasmid mini-preparation (SPH)
 
  1. Grow a colony in 5 ml LB + antibiotic overnight at 37°C in a Falcon tube (orange cap).  Use 100 mg/ml ampicillin for overnight culture.
  2. Spin at 3K for 5 min.  Discard supernatant.
  3. Resuspend in 1 ml STE buffer.  Transfer to a 1.5 ml microfuge tube.
  4. Spin at 5K for 5 min.  Discard supernatant.
  5. Resuspend by vortexing in 200 ul GTE + lysozyme buffer.  Incubate at room temp. for 5 min.
  6. Add 400 ul fresh NaOH/SDS buffer.  Mix by inverting.  Incubate on ice for 5 min.
  7. Add 300 ul potassium acetate buffer.  Vortex.  Incubate on ice for 5 min.
  8. Spin at 14K for 5 min.  Move supernatant to a new microfuge tube.
  9. Add 200 ul chloroform and 200 ul phenol.  Vortex.
  10. Spin 14K for 5 min.  Move the UPPER PHASE to a new microfuge tube.  Avoid material at the interface.
  11. Fill microfuge tube with isopropanol.  Mix by inversion and vortex.  Incubate at room temp. for 2 min.
  12. Spin at 14K for 5 min.  Discard supernatant.
  13. Add 1 ml ice-cold 70% ethanol.  Invert a couple of times.
  14. Spin at 14K for 5 min.  Discard supernatant.
  15. Vacuum dry or spin in the Speed-Vac to remove remaining liquid.
  16. Resuspend in 50 ul TE buffer.
 


STE buffer

0.1M NaCl, 10 mM Tris-HCL pH 8, 1 mM EDTA


GTE buffer

50 mM glucose, 25 mM Tris-HCL pH 8, 10 mM EDTA

Add lysozyme to 4 mg/ml


NaOH/SDS solution

0.2M NaOH, 1% SDS


Potassium acetate buffer

3M potassium acetate      60.0 ml of 5M
2M glacial acetic acid      11.5 ml
water                                 28.5 ml

Mutagenesis

1 ul template DNA (~50ng)
5 ul 10X PCR buffer
1.25 ul forward oligo (100 ug/ul)
1.25 ul reverse oligo (100 ug/ul)
5 ul dNTP's (1.5 mM)
1 ul Turbo enzyme
32 ul water
50 ul total

Typical scheme:

25-35 cycles - 1 min at 94°C, 1min at 60°C, 14 min at 72°C

* After PCR, add 1 ul DpnI to the mixture and incubate for 1 hour at 37°C to destroy the template DNA.  Purify from an agarose gel.