Protocols

Preparation of HNF3g YAC DNA for Microinjection

Courtesy of Newman Sund and Dr. Klaus H. Kaestner, Department of Genetics, University of Pennsylvania School of Medicine (For additional reference: Schedl, A. et al. 1993 Nucleic Acids Research. 21: 4783-4787.)

[Large Scale Preparation of Yeast Plugs] [Solutions]
  1. Equilibrate 8 yeast plugs (Large Scale Yeast Plug Prep) 1x 10', 1x 30', 1x 1hr in 50 ml/wash of 0.40x TBE (BioRad 10xTBE #161-0733). Equilibrate "PFGE markerI-Lambda ladder"(BMB #1378961) at this time as well. It is recommended that you make 3.0 liters of 0.40xTBE for plug washes, gel and running buffer.
  2. Load plugs into 4 cm. slot of 1.0 % BioRad PF-certified agarose gel in 0.40xTBE. Make a 200 ml gel using the preparative comb. Plugs are positioned vertically, tightly next to each other, and flanked by l ladder. Seal the plugs in the gel with 1.5% SeaPlaque Low Melting Point (LMP) agarose.

    Note
    - The original protocol uses a 1.0% SeaPlaque (FMC) LMP agarose PF gel. Non-LMP agarose is recommended for use because the 1% LMP agarose gel is fragile and difficult to work with in steps 5 - 7. When excising the YAC DNA in step 8 using the non-LMP agarose, make sure not to excise the 1% gel with the 4% gel. The non-LMP agarose will not melt at 68°C in step 10. Using the non-LMP agarose may affect the migration of the YAC DNA into the 4% LMP gel and affect the yield.

  3. Run gel as optimized previously. For HNF3g YAC: 5-15 s. switch, 20 hr. run, 6V/cm, 120° angle, 14°C, 2.4 L of 0.40xTBE. You want the current to run 120-145 amps during the run, closer to 120A at the beginning of the run.
  4. After the run, do NOT stain the entire gel. Instead, cut off both sides of the gel (l ladder plus half of one lane each - see Figure 1) and stain the marker gel pieces in 0.5 mg/ml Ethidium Bromide (EtBr) (20 mL 10 mg/mL EtBr stock in 400 mL dH20) for 30 min. rocking. While marker gel pieces are staining, soak rest of the unstained gel in 1 x TAE buffer (Maniatis). It is recommended that 4L of 1 x TAE is made for all the washes and the running buffer. Observe marker gel pieces under UV and take picture of gel with ruler aligned with the top of the gel. Note the position of the YAC band as well as two reference chromosomes.
    Figure 1Figure 1
  5. Now excise the YAC band and the two individual reference chromosomes from the unstained, pulse field gel (cut out ±2.5mm). Stain the rest of the gel to confirm that you cut out the correct band.
  6. Position gel slices in a minigel tray with the YAC slice in the middle (see Figure 2). The gel slices should be positioned LENGTHWISE, such that the DNA will migrate at a 90° angle to the original PFGE run. Seal the sides of the gel pieces onto the tray with 4% LMP-agarose (NuSieve GTG, FMC) in 1x TAE to keep the gel pieces precisely positioned. Prepare the 4% LMP gel beforehand and keep at 48°C. LMP agarose foams and boils over easily so watch it carefully as it heats. Cast 4% LMP-agarose in 1 x TAE around the slices, barely covering them (~180-200 mL). Make sure that the surface is level when casting and running the gel. Keep some tape on tray ends to prevent the gel from sliding off during the run. Electrophorese for 16 hours at 60V (2.2V/cm) while recirculating the buffer.
    Figure 1Figure 2
  7. Excise one entire reference chromosome plus at least 3 cm into the 4% gel where the DNA has migrated into and stain the gel piece in EtBr, as above. If there is still a significant amount of DNA in the 1% gel, electrophorese longer at 110V (4V/cm) for an additional 1-2hr and stain the other reference chromosome. Estimate the position of the YAC based on the position/location of the reference chromosome DNA and cut out the minimal YAC band. Place into tared mfuge tube and determine volume of the block. 1 mg =1 mL. If necessary, cut the agarose block into smaller pieces such that each block is approximately 100-200 mg. This aids melting in step 10 and potentially concentrates the DNA in some aliquots. Stain the rest of the YAC slice to confirm you cut it out.
    Figure 1Figure 3
  8. Equilibrate gel block 1x 10', 2x 1hr. in 15 ml/wash of 1 x TAE, 100 mM NaCl, 30 mM Spermine, 70 mM Spermidine.
  9. Melt agarose block for 20 min at 68°C, stirring the solution after 10 minutes with a sterile pipette tip. Cut the gel block into smaller pieces to aid melting. Do not incubate agarose at temperatures greater than 70°C, as it will denature the DNA. Once melted, transfer tube immediately into 42°C water bath and equilibrate the molten agarose for 10 minutes at 42° C. GELase loses activity at temperatures greater than 45°C. Add 5 units (1U/mL) GELase (Epicentre) per 100 mg agarose and incubate at 42°C for 2 hours. While in heat block, stir molten agarose a couple of times with pipette to ensure even distribution of GELase. After 2 hours, place test tube on ice for 10 minute to test if digest is complete. If gel reforms, repeat step 10 with more GELase.
  10. Pulse briefly at 3000 rpm for 10 sec. Dialyze against YAC micro-injection buffer for 3 hours. For this, place a Dialysis Membrane (0.05 microns, type VM, Millipore) on top of 50 ml of YAC micro-injection buffer in a Petri-dish. Pipette YAC DNA solution carefully on top of the membrane using a cut-off blue tip, dialyze for 3 hours, and transfer YAC DNA to a fresh mfuge tube. Save a 1 ml aliquot of YAC micro-injection buffer for dilution of YAC DNA in step 13.
  11. Estimate YAC DNA amount by PFGE (load 20 mL) against known standards (10, 20, 40, and 80 ng). Include positive control and l marker as well. Can stop run at 18.5 hours to see plasmid stds.

    Notes for handling YAC DNA
    - Always tap mfuge tube to mix purified YAC DNA prior to taking off an aliquot.
    - Never vortex or pipette up and down to mix. This will shear the linear YAC DNA.
    - Use a cut-off pipette tip when you need to take an aliquot of YAC DNA.
    - Store YAC DNA at 4°C until ready for use.

  12. Dilute YAC DNA to 0.3-2.0 ng/mL. You have to dilute at least 2:1 with YAC micro-injection buffer: YAC DNA for micro-injection. Therefore, the YAC DNA must be at minimum 0.9 ng/mL when quantitated.


    YAC micro-injection buffer
    (10 mM Tris/Cl pH 7.5, 0.1mM EDTA pH 8.0, 100 mM NaCl, 30 mM Spermine, 70 mM Spermidine)

    For 200 ml
    1M Tris pH 7.5 2.0 mL
    5M NaCl 4.0 mL
    0.5 EDTA 40 mL
    0.3M Spermine (1:10,000 diln) 20 mL
    0.7M Spermidine 20 mL
    MQ ddH2O 194 mL
    1xTAE, 100 mM NaCl, 30 mM Spermine, 70 mM Spermidine
    For 200 ml
    50xTAE 4.0 mL
    5M NaCl 4.0 mL
    0.3M Spermine 20 mL
    0.7M Spermidine 20 mL
    MQ dd H2O 192 mL
    0.3M Spermine (348 g/mol)
    For 1 ml: 0.104g in 1.0 ml ddH20
    0.7M Spermidine (255 g/mol)
    For 1 ml: 0.179 g in 1.0 ml ddH20

Large Scale Preparation of Yeast Plugs

  1. Inoculate 100 ml medium (YPAD or selective medium) with 0.2 to 1 ml of a stationary culture. Shake at 30°C for 1 to 2 days. Density should be at least 5 x 107 cells/ml.
  2. Pellet cells in 50 ml Falcon tubes at 3000 rpm for 5 minutes.
  3. Resuspend pellets in 40 ml (total) 50 mM EDTA, pH 8. Spin as above.
  4. Weigh a Falcon tube. Resuspend cells in Solution 1 and pellet in Falcon tube.
  5. Determine the weight of the pellet. Assuming 1g = 1ml, you now know the volume of the pellet. Add 0.5 volume Enzyme Solution and warm to 37°C.
  6. Now add equal volume of pre-warmed (40°C) Agarose Solution. Suspend the cells using a cut-off blue tip. Pipett into 8- microliter block forms.
  7. Place on ice for 10 min. Gently remove plugs and incubate for 2 hours at 37°C in 5 ml per plug of Solution 2.
  8. Transfer to LDS at 37°C. After one hour, transfer to fresh LDS and incubate (gentle shaking) at 37°C overnight.
  9. Next day, wash plugs for two hours in 20% NDS at room temp. Store plugs indefinitely at room temperature.
  10. Before use, plugs have to be equilibrated in restriction buffer or TE.

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Solutions

Solution 1
1 M Sorbitol
20 mM EDTA, pH 8
14 mM b-mercaptoethanol (b-ME)

Solution 2
1 M Sorbitol
20 mM EDTA, pH 8
14 mM b-ME
10 mM Tris/HCl pH 7.5
2 mg/ml Zymolase-20T (ICN)
(make up fresh)

Enzyme Solution
6 mg/ml zymolase-20T (ICN) in Solution 1

100% NDS
350 ml H2O, 93 g EDTA
0.6 g Tris base.
1. Add NaOH pellets until pH > 8.0.
2. Dissolve 5 g N-Lauryl-Sarcosine in 50 ml H2O and add to above.
3. PH to 9.5 with NaOH, adjust volume to 500 ml.
4. Store at room temperature.

Agarose Solution
1 M Sorbitol
20 mM EDTA, pH 8
2% Sea Plaque GTG Agarose (FMC)
Once melted, cool to 40 C and add b-ME to 14 mM

LDS
100 mM EDTA pH 8.0
10 mM Tris/Cl pH 8
1% Lithiumdodecyl sulfate

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