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Kansas State University

Plasmid and BAC DNA Isolation Protocol

  • Inoculate a single colony in 5 ml of LB plus suitable antibiotics and grow 16 to 20 hr at 37oC.
  • Pipette 170 ml of the overnight culture into a 500 μl tube that contains 30 μl glycerol. Mix it and store at –70oC.
  • Centrifuge the rest of the culture at 1,500g in a desktop centrifuge for 10 mins, pour out the supernatant, resuspend the pellet in the remaining culture (left over) with a Votex. Add 0.2 ml of solution I [containing 50 mM glucose, 10 mM EDTA, and 25 mM Tris (pH 8.0)] and incubate on ice for 5 mins.
  • Add 0.4 ml of freshly prepared solution II (0.2 N NaOH and 1% SDS), mix gently, and incubate on ice for 5 mins.
  • Add 0.3 ml of solution III (3 M Kac, pH 5.3 - 5.5), mix gently, and freeze at - 80 C for 15 mins to overnight.
  • Thaw at room temperature and centrifuge at 2,800g in a desktop centrifuge for 15 mins in a microfuge to collect the precipitate.
  • Transfer 0.8 ml of the supernatant to a new microtube. Add 2 ml RNase and incubate 1 to 3 hours.
  • Add 0.5 ml of Phenol/chloroform, vortex, and centrifuge for 5 min.
  • Transfer 0.7 ml supernatant into a new tube that contains 0.7 ml isopropanol, mix, and centrifuge at 12,000g in a microcentrifuge for 5 mins to pellet DNA.
  • Remove the supernatant, wash the pellet with 70% ETOH, and centrifuge at 12,000g for 2 mins.
  • Briefly centrifuge and remove the remaining ethanol solution by pipetting.
  • Dissolve the pellet in 50 ml H2O.
  • Pipette 5 ul of the DNA solution into a tube containing 5 ml H2O and 1 ml 10 x TAE loading buffer. Store the rest of the DNA solution in –70oC.
  • Analyze the DNA in loading buffer on 1% agarose gel.

Solution I: (100 ml) 
Glucose 0.9g 50 ml 
EDTA (pH 8.0) 2 ml of 0.5 M stock 10 ml 
Tris-HCl (pH 8.0) 5 ml of 1 M stock 25 ml

Solution II: NaOH-SDS stock 
Ingredient Amount Final Concentration
NaOH 1/2 volume of 0.4 N stock 
(8g/500 ml) 
0.2 N SDS 1/2 volume of 2% stock 
(10g/500ml)
1% 
*Adjustment: Mix NaOH and SDS stocks just prior to use.

Solution III: 
Potassium acetate stock (pH 4.8-5.3): (100 ml) 
Potassium Acetate 60 ml of 5 M KOAc 
Glacial Acetic Acid 28.5 ml 
H2O 11.5 ml 

BCA Protein Assay Protocol: Before starting the assay, get samples (10 ml of each in individual tubes) and BSA standard and put them on ice. Turn on the incubator and set it to 60oC. Be sure to use autoclaved water.

Prepare a series of standards:

Label 5 micro-tubes 5, 10, 20, 40, and 80, respectively. 
Pipette 5, 10, 20, 40, and 80 ml of the BSA standard into each of the above tubes.
Add, 75, 70, 60, 40, and 0 ml of water into the above tubes, respectively.
Add 10 ml of sample extraction buffer into each tube.

  • Prepare samples: Add 80 ml of water into each sample tube.
  • Prepare BCA working reagent: For 10 testing samples including standards, pipette 10 ml of reagent A into a 50 ml tube. Pipette 200 μl of reagent B into the same tube that contains A and mix them.
  • Conduct the assay: Add 1 ml of the BCA working reagent into each BSA standard, blank, and sample tubes. Incubate all tubes in a 60oC water bath for 15 minutes and in a RT water bath for 5 minutes. Measure the absorbance of the solutions at 562 nm. Calculate protein concentrations of samples according to the standard curve.

Plant Protein Extraction:

  • Grind Plant tissue in liquid nitrogen to a fine powder.
  • Add 2 volumes of extraction buffer and grind the tissue sample more (Optional: Sonicate the sample for 15 sec with microprobe).
  • Spin at 13,000 rpm for 30 min in a Microfuge at 4oC
  • Transfer supernatant into a new clean tube
    Pipette 10 ul for determination of protein concentration
    Store the rest in -80 oC.

Extraction Buffer:

100 mM Hepes, pH7.5
5 mM EDTA
5 mM EGTA
10 mM DTT
10 mM Na3VO4
10 mM NaF
50 mM b-glycerophosphate
1 mM phenylmethysulfonyl fluoride
5 ug/ml antipain
5 ug/ml aprotinin
5 ul/ml leupeptin
10% glycerol
7.5% polyvinylpolypyrrolidone

Preparation of SDS/PAGE:

  • Wash glass plates, spacers, and combs
  • Wipe the above items with 95% Ethanol and set up them in a gel making box.
  • Prepare 10% SDS/PAGE separating gel solution:           
    Seal the bottom and sides of the gel sets
    Pour the gel solution into the gel sets until it reaches about 1 inch below 
    the edge of the lower plate
    Overlay with Butanol (water satuated) and let it polymerize for at least 40 min
  • Make stacking gel with the following mix: 
  • 8 ml Water, 4 ml Upper gel buffer, 2.4 ml Acrylamide (30%), 2.4 Bis, 
    200 ul 10% APS, and 10 ul TEMED

Suck up Butanol with filter papers from the separating gel
Pour the stacking gel mix into the gel sets
Put a comb into each gel set and let the polymerize for 20 min.

Mix protein extract (20 ug of total proteins) with 4 ul 10x Loading buffer and  
water (total vol 40 ul)
Boil the sample mix for 10 min
Microfuge and load the sample mix onto the gel

Run the gel at 60 mA for 2 – 3 hours.

Buffers:
1. Sample Buffer (10 ml):1 ml Glycerol, 0.5 ml 2-mercaptoethanol,0.23 g SDS, 0.08 g Tris, pH 6.8.
2. Running Buffer (1 L):30.3 g Tris base, 144.0 g Glycine, 10.0 g SDS, pH 8.3.
3. 10 x PBS (Phosphate-buffered saline) (1 L):80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4, pH 7.4.

Gel Kinase Assay :

  • Wash the gel 3 times with buffer A after electrophoresis (30 min for each at RT)
  • Incubate the gel in buffer B at 4oC overnight with three time buffer changes
  • Incubate the gel in buffer C for 30 min at RT
  • Incubate the gel in 10 to 20 ml buffer D containing 200 nM ATP plus 1.85 mBq g-32P-ATP for 9 min
  • Incubate the gel in buffer D for 3 hours with at least 5 changes of the same solution.
  • Dry the gel on 3 mm filter paper
  • Expose the dried gel to X-ray film or analyze on a PhosphorImager.
  • Buffer A:
  •  25 mM Tris-HCl, pH7.5, 0.5 mM DTT, 0.1 mM Na3VO4, 5 mM NaF, 0.5mg/ml BSA and 0.1% Triton X-100
  • Buffer B:
  • 25 mM Tris-HCl, pH7.5, 1 mM DTT, 0.1 mM Na3VO4, 5 mM NaF
  • Buffer C:
  • 25 mM Tris-HCl, pH7.5, 1 mM DTT, 0.1 mM Na3VO4, 2 mM EGTA, 12 Mm MgCl2
  • Buffer D:
  • 5% trichloroacetic acid and 1% sodium phosphate

Plasmid Size Determination:

  1. Inoculate Bacteria and incubate overnight. (Care should be taken to avoid cross contamination by misplace the inoculators and to avoid picking up un-thawed ice from the original plate)
  2. Transfer the bacterial culture from the plate into individual tubes (Maker sure the numbers on the plate match exactly the numbers on the tubes.)
  3. Centrifuge and remove the supernatant.
  4. Add 30 µl TE into each tube and vortex to dissolve the bacterial precipitates.
  5. Add 30 µl Phenol/Chloroform and Vortex. (Wear glass and lab coat.)
  6. Centrifuge.
  7. Transfer 25 µl of the uplayer solution into small tubes that contain sample buffer. (Again, make sure the numbers on the big tube match exactly the numbers on the small tubes.)
  8. Make an 1% agarose get.
  9. Load the samples into the wells in the gel and run the gel with 120 V for 3 hours.
  10. Take a picture using the BioDoc system.
  11. Compare the sizes of individual clones and record the results on the log book. Attack the picture to the log book for later reference
Southern Blot Analysis:
  1. After electrophoresis is completed, photograph the gel.
  2. Remove any unnecessary part of the gel. Mark the markers’ positions using a needle with Indian ink.
  3. Denature the DNA by soaking the gel in several volumes of 1.5 M NaCl/0.5 M NaOH for 15 min.
  4. Meantime, prepare a stack of whatman 3 MM paper cuts (larger than the gel size), put this set of paper cuts into a large baking dish, soak the paper cuts with 1.5 M NaCl/0.5 M NaOH solution.
  5. Invert the gel so that its original underside is not uppermost. Place the gel onto the damp 3MM paper cuts (make sure there is no air bubbles) between the paper cuts and the gel).
  6. Cut a piece of GeneScreen membrane (same size as the gel), mark the coated side with pencil, and wet the membrane in water for 5 min.
  7. Place the membrane onto the gel (with the coated side facing the gel, no air bubbles between the gel and the membrane).
  8. Wet two pieces of Whatman paper (same size as the gel) and put them onto the membrane individually.
  9. (Optional: Use Sara-rapper to cover the baking dish, and remove the Sara-rapper within the gel region with a razor)
  10. Put a stack of paper-towels (same size as the gel) on top of the Whatman papers.
  11. Put a glass plate on top of the stack together with a 500 g weight.
  12. Let it transfer overnight (be sure there is enough solution, but not exceed the level of the gel).
  13. Remove the towels and the Whatman papers. Turn over the dehydrated gel and together with the membrane and lay them on a dry sheet of Whatman paper. Mark the positions of the gel slots and the DNA markers on the membrane with a soft pencil.
  14. Peel off and discard the gel. Soak the membrane in 6 x SSC/200 mM Tris, pH7.5 buffer for 5 min.
  15. The filter is now ready for hybridization.