4X Tris�Cl/SDS pH 8.8 Buffer

• 91 g Tris base

• 300 mL H₂O

• Adjust to pH 8.8 with HCl

• Add H₂O to 500 mL total volume

• 2 g SDS

4x Tris�Cl/SDS pH 6.8 Buffer

• 6.05 g Tris base

0.4 g SDS

• 40 mL H₂O

• Adjust to pH 6.8 with HCl

• Add H₂O to 100 mL total volume

30% acrylamide/0.8% bisacrylamide

• Store at 4°C in the dark

• 30.0 g acrylamide

• 0.8 g N, N'-methylenebisacrylamide

• Add H₂O to 100 mL total volume

5x SDS Electrophoresis Buffer

• 15.1 g Tris base

• 72.0 g glycine

• 5.0 g SDS

• Add H₂O to 1000 mL total volume

Isopropanol Fixing Solution

• 25% (vol/vol) isopropanol

• 10% (vol/vol) acetic acid

Rapid Coomassie Staining Solution

• 10% (vol/vol) acetic acid

• 0.006% (wt/vol) Coomassie G-250

Destaining Wash Solution

• 10% (vol/vol) acetic acid

Protocol:

1. Make fresh 10% Ammonium Persulfate.

2. Assemble the gel casting apparatus, making sure that the sandwich of glass plates and spacers will make a good seal.

3. Prepare the Separating Gel solution according to the acrylamide concentration needed. Vortex.

Separating Gel

1. Load the apparatus with 4.5 mL of the Separating Gel solution.

2. Top with ~1 mL of Isoamyl alcohol to isolate the polymerization from oxygen.

3. After polymerization, pour off the Isoamyl alcohol, and rinse with distilled water.

4. Remove any water droplets from the inside of the casting apparatus with Whatman paper or a paper towel. Insert the comb for the stacking gel.

5. Prepare the Stacking Gel solution. Vortex.

Stacking Gel (5% acrylamide)

1. Load the Stacking Gel solution, taking care not to introduce air bubbles around the comb (some bubbles can be removed by pipetting up and down).

2. Allow the Stacking Gel to polymerize completely (~45 minutes) before removing comb.

3. Prepare the samples:

   1. Dilute the protein sample 1:1 with 2x SDS Sample Buffer.

   2. Heat the samples and the molecular weight standards for 5 minutes at 100°C.

4. Remove the glass and gel sandwich from the casting apparatus.

5. Clip the sandwich to the electrophoresis apparatus. Carefully remove the comb from the gel and fill the top of the apparatus with 1x SDS Electrophoresis Buffer.

6. Using a 20-gauge needle, flush the wells with buffer.

7. Carefully load the samples into the bottom of the wells using a flat-tipped pipette tip.

8. Fill the bottom of the electrophoresis apparatus with 1x SDS Electrophoresis Buffer and connect the apparatus to the power supply.

9. Run the gel at 10 mA until the dye enters the separating gel. Then increase the current to 15 mA.

10. When the dye reaches the bottom of the separating gel, turn off the power supply, and remove the gel sandwich.

11. Carefully open the sandwich by using one of the spacers to pry the plates apart.

12. Gently cut away the stacking gel and place the separating gel in a small plastic container for staining.

13. Cover the gel with fixing solution and shake gently for 15 minutes.

14. Pour off the fixer and cover the gel with staining solution. Shake gently for at least 2 hours.

15. Pour off the staining solution and cover the gel with the wash solution. Destain for at least 2 hours. (It is usually necessary to change the wash solution at least once)

16. The gel can be stored in water or dried down between sheets of cellulose on a drying frame.

Angela McKay, 22 December 1999

1. ONE ORDER = ONE PLATE = 96 TOTAL SAMPLES ….. (there should be no empty wells).

2. Samples are to be loaded in vertical columns going from A-1 to H-1, A-2 to H-2, A-3 to H-3 and so on till A-12 to H-12 .

3. We must receive the full 12 �l volume of ‘DNA + primer + dI water’ cocktail.

4. Since the sample volume is small, the plates must be ‘V’ bottomed, 200 �l capacity (not 500 �l), stable on bench-top, and well-sealed to withstand cross contamination between wells in transportation from Rosen to Hitchcock.

5. The plates must be CLEARLY LABELLED WITH THE CORRESPONDING ORDER NUMBER. ORDER NUMBERS ARE UNIQUE NUMBERS GENERATED BY THE SOFTWARE. If instead of order numbers, plates bear #1, #2, OR ‘forward,’ ‘reverse,’ etc., THEY WILL NOT BE PROCESSED.

6. Being ‘high throughput’ and fast, we do not want to be tethered to the client by phone/by e-mail following each sample drop-off. Please take the time in your lab to MATCH THE PAPER-WORK WITH SAMPLES. If you are “too rushed” …. STOP …. keep the samples with you!

7. When new users from your group want to use our facility… please, work with them a few times until they are comfortable with these guidelines.

Dec 7, 2004

• Big Dye Terminator kit from Perkin Elmer

• Gene specific primers (need 3.2 pmol/reaction)

• Sample DNA:

  • 200-300 ng for dsDNA
  • 75-100 ng for ssDNA
  • 90-150 ng for PCR products

• ddH₂O

• Isopropanol

• Mineral Oil

Protocol:

1. Quantify DNA or PCR product using the Spectrophotometer. The protocol applies to a full reaction. In case of a half reaction, divide all volumes in half but DO NOT decrease DNA or primer amounts.

2. Mix sample DNA and specific primers to 12 µl total volume.

3. Add 8 µl Terminator Ready Reaction mix.

4. Mix well and spin briefly (do not vortex).

5. If thermal cycler does not have a heated lid, overlay the reaction mix with 40 µl mineral oil.

6. Put the tubes in thermal cycler and set volume to 20 µl.

7. Program cycle below and repeat for 25 cycles:

   1. In a GeneAmp 9600/9700 or 2400

      1. 96°C for 10 seconds

      2. 50°C for 15 seconds

      3. 60°C for 4 minutes

      4. 4°C soak until needed

   2. In a Thermal Cycler (TC1) or in a DNA Thermal Cycler 480

      1. 96°C for 30 seconds

      2. 50°C for 15 seconds

      3. 60°C for 4 minutes

      4. 4°C soak until needed

8. Spin for 1 minute to remove condensation.

9. Add 20 µl ddH₂O and 60 µl 100% Isopropanol, for 100 µl final volume. (Half reactions: add 30µl ddH₂O instead of 20 µl.)

10. Precipitate at room temperature for 20-30 minutes.

    1. Strip well or 96-well plates:

       1. Spin for 30 minutes at 3000x g

       2. Remove from centrifuge and turn upside down on paper towel

       3. Spin for 1 minute at 700x g to remove residual Isopropanol

       4. Remaining Isopropanol: dry in 37°C oven for 2 minutes (no longer).

    2. Individual tubes:

       1. Spin for 20 minutes at 14000 rpm

       2. Remove the Isopropanol after spinning

       3. Add 250 µl 75% Isopropanol, vortex briefly

       4. Spin for 5 minutes at 14000 rpm

       5. Remove Isopropanol.

       6. Dry in heating block.

11. Fill out sequencing data form.

12. Put the samples in the -20°C freezer by the DNA Sequencing facility.

Lysis buffer:

0.45% Tween, 0.45% NP40, 2.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-Cl pH8.3, 100 µg/µl, Proteinase K

* Add Proteinase K just before use, 5 µl stock per ml of lysis buffer used.

A. Protocol:

1. Wash cells with cold PBS.

2. Resuspend at 10,000 cells/µl in lysis buffer.

3. Incubate at 56°C for 1 hour.

4. Boil for 10 minutes.

5. Store lysate at -20°C

B. Reagents:

L6 buffer:

0.08 M GuSCN (LifeTechnologies GibcoBRL), 0.08 M Tris-Cl pH 6.4, 0.035 M EDTA, 2% (wt/vol) Triton X-100

B. Protocol^*:

1. Wash cells with cold PBS

2. Resuspend at 10⁶ cells/ml in L6 buffer.

3. Add an equal volume of Isopropanol.

4. Spin at 14000 rpm for 30 minutes at 4°C.

5. Discard supernatant.

6. Wash pellet twice with 75% Ethanol.

7. Airdry pellet.

8. Resuspend pellet in 100 µl dH₂O. Store at -20 °C.

* Adapted from Boom et al. J.Clin.Microbiol. 1991, 28:495-503.

• DNA-free^TM, 50 reactions, Ambion #1906, $65

• Oligo dT, 24mer (prep’d by Sherry)

• dNTP Set, 4 x 25 µmol 100 mM, Bioline #BIO-39025, $125

• RNase Inhibitor, 2,000 U, Roche Applied Science #3335399

• SuperScript III Reverse Transcriptase, 2,000 U, Invitrogen #18080-093, $59.40

• Ribonuclease H, 30 U, Invitrogen #18021-014, $99

• TaqMan^� beta-actin Detection Reagents, 100 reactions, ABI #401846, $150

• TaqMan^� Universal PCR Master Mix, No AmpErase^� UNG, 200 reactions, ABI #4324018, $355

Protocol:

DNA digestion:

1. Keep extracted RNA on ice or thaw total RNA on ice (10 pg- 5 µg required total RNA).

2. Thaw DNA-free reagents on ice.

3. Add 0.1v 10 x DNase buffer and 1 µl DNase I to appropriate amount of total RNA and mix by flicking.

4. Incubate 20 minutes at 37°C.

5. Mix DNase Inactivation reagent well. If reagent is difficult to mix, add 1v H₂O/0.1 mM EDTA.

6. Add 0.1v DNase Inactivation reagent.

7. Incubate 2 minutes, flicking intermittently.

8. Spin 1 minute at 10,000 x g to pellet DNase Inactivation reagent.

9. Remove supernatant and use, or store at -70°C.

cDNA synthesis:

1. Keep on ice: oligo DT (20 mM), dNTP (20 mM), 5x SST buffer, DTT, SuperScript III, RNase Out, and DNA digested RNA.

2. To 4 µl DNA digested RNA, add 8 µl of the following mastermix for 1 reaction(s) (enter # of reaction):

   • 1 µl oligo DT (20 µM)
   • 0.5 µl dNTP (20 mM)
   • 6.5 µl ddH₂O

3. Incubate 5 minutes at 65°C.

4. Cool on ice.

5. While cooling, make the following mastermix for 1 reaction(s) (enter # of reaction):

   • 4 µl 5x 1st strand buffer
   • 2 µl 100 mM DTT
   • 1 µl RNase OUT (RNase inhibitor)

6. Add 7 µl of mastermix.

7. Add 1 µl of SSTIII (SuperScript III). Flick and collect.

8. Run sample in thermocycler:

   1. 42°C for 50 minutes

   2. 70°C for 15 minutes

   3. 4°C soak until needed

1. Add 1 µl RNase H to remove complimentary strand.

2. Incubate 20 minutes at 37°C.

3. Estimate cDNA yield using a spectrophotometer.

4. Dilute to appropriate concentration: 5 µl TaqMan template input.

TaqMan:

1. Aliquot 5 µl sample and/or DNA template to optical tubes. Seal with Parafilm.

2. Prepare Mastermix in PCR setup room. Click to download Mastermix Wizard

3. Add 15 µl Mastermix to optical tubes and cap. Keep on ice and transfer to ABI 7700.

4. Flick and spin to collect. Run on ABI 7700.

1. Add 0.7 - 1.0 U of Taq polymerase (Biolase) to PCR product.

2. Mix well. Incubate at 72 °C for 8-10 minutes. Use immediately.

Modified Topo T/A cloning protocol

Preparation:

1. Spread each Carb. plate with 40 µl of 40mg/ml X-gal.

2. Pre-warm plates in warm room.

Ligation:

1. Dilute PCR product 1 in 4 with dH₂O.

2. Combine:
   Topo T/A vector mix 0.25 µl
   PCR product dilution 1.00 µl
   dH₂0 1.25 µl

3. Incubate 5 minutes at room temperature, and then put on ice.

Transformation:

1. Top10 cells should be thawed GENTLY and placed on ice; keep everything cold.

2. Combine:
   Ligation 1.5 µl
   0.25ul β-ME 1.0 µl
   Top10 cells 10.0 µl

3. Stir gently with pipette tip.

4. Chill on ice for 15 minutes.

5. Heat shock in 42°C water bath for 30 seconds.

6. Chill on ice for 2 minutes.

7. Add 125 µl SOC-media and incubate -shaking- at 37°C for 1 hr.

8. Plate the entire transformation on LB-carb + X-Gal plates.

9. Place plates in warm room overnight.

10. When cloning a 750 bp product, this procedure consistently gives 100-200 colonies per plate.

RNA Extraction (QIAgen Viral RNA Kit) (BL3)

When working in the BL3, remember to use extreme care. Clean the workspace with 70% Et-OH before and after use, and make sure that contaminated tubes and tips are neutralized with Westcodyne.

• Check the heat block. Heat to 80°C, if needed.
• Pull the samples from the freezer to thaw.

Buffer AVL precipitates at 4°C after addition of the Carrier RNA.

• Remove Buffer AVL from the fridge and place on the 80°C heating block. Vortex after ~2 minutes. Do not heat for more than 5 minutes, and do not heat more than 6 times.

Carrier RNA dissolved in buffer AVL is stable for up to 6 months at 4°C.

For each quadruple extraction:

• 1 5mL tube to contain the buffered plasma
• 2-4 cryo tubes for the extra plasma aliquots
• labels for the aliquots
• 10 collection tubes
• 1 1.5mL tube
• 1 spin column (in a collection tube)

Before using buffer AVL for the first time, check the buffer for precipitate, and incubate at 80°C, if needed, until precipitate dissolves. Add 1 mL of buffer AVL to the tube of lyophilized Carrier RNA. Dissolve the RNA thoroughly and transfer back to the buffer bottle. Mix thoroughly and aliquot 1.0mL into dated 1.5 mL tubes. Store at 4°C and use within 6 months.

Before using buffers AW1 and AW2 for the first time, add the appropriate amount of Et-OH. Mix thoroughly.

1. All buffers should be at room temperature prior to use.
2. Label the 5mL tubes.
3. Pipet 2240μL of Buffer AVL into each 5mL tube.
4. Add 560μL of the plasma sample. Mix thoroughly by pipetting. (some samples foam at this point)

If cryoprecipitates are present in the thawed plasma sample, they can be pelleted by briefly centrifuging at 6800 x g for 3 minutes. This step will not reduce viral titers.

1. Incubate at room temperature for 10 minutes.
2. Aliquot the remaining plasma, label tubes, and place in freezer.
3. After 10 minutes, add 2240μL 96% Et-OH to the 5mL tube. Mix thoroughly.
4. Remove 630μL from the 5mL tube and apply it to the spin column. Take care to not wet the rim.
5. Spin at 6000 x g for 1 min.
6. Place spin column in a clean collection tube and discard the tube containing the filtrate.
7. Repeat the previous three steps until all the buffered plasma has been applied to the column.
8. Label the 1.5mL tubes.
9. Wash the column with 500μL of Buffer AW1.
10. Spin at 6000 x g for 1 min.
11. Place spin column in a clean collection tube and discard the tube containing the filtrate.
12. Wash the column with 500μL of Buffer AW2.
13. Centrifuge at full speed for 3 minutes. (20,000 x g)
14. Place spin column in a clean collection tube and discard the tube containing the filtrate.
15. Centrifuge at full speed for 1 min.
16. Place the spin column in the labeled 1.5mL tube. Apply 40μL of Buffer AVE directly to the center of the membrane and incubate at room temperature for 1 min.
17. Centrifuge at 6000 x g for 1 min.
18. Apply another 40μL of Buffer AVE to the center of the membrane and incubate at room temperature for 1 min.
19. Centrifuge at 6000 x g for 1 min.

Place the 1.5mL tubes on foil and spray with 70% Et-OH. Make a packet with the foil and spray the outside with 70% Et-OH before removing from the BL3.

In the PCR room, aliquot 25μL of the RNA into labeled 0.6mL tubes using low retention tips.

Viral RNA is stable for up to 1 year when stored at -20°C or -70°C.

Proceed to cDNA reaction for one aliquot immediately following extraction. This avoids a freeze/thaw of our sample and may allow a greater chance of successful amplification.

Whole Genome Reverse Transcription (Invitrogen RT Kit)

1. Mix per sample and preheat RT-PCR machine to 65°C:

   	RNA	~25μL
   	dNTP (20mM)	1.5μL
   	oligo-dT (20pmol/μL)	2.5μL

2. Start file#33 - heat at 65°C for 5 min
3. Reduce heat to 45°C (higher temp will inactivate RT!)
4. Add 20μL of the following pre-warmed!!! mix per sample:

   	5x buffer	8μL
   	0.1M DTT	4μL
   	Superscriptase III	2μL
   	RNase inhibitor	1μL
   	dH2O	5μL

5. Incubate 1.5 hours at 45°C
6. Add additional 1.0μL Superscriptase III, and incubate another 1.5 hours at 45°C
7. Inactivate for 15 min at 70°C
8. Add 1μL RNase H and incubate for 20 min at 37°C
9. Label tube as cDNA and date. Freeze in Walker cDNA box.

Hot Start PCR

dNTPs final concentration: 360 μM

primers final concentrarion: 300nM

MgCl final concentration = 1.75 mM

Cycle Profile: WHOLGENM

SET-UP (individual tubes and paraffin):

1. Place 1mL microfuge tube filled with paraffin in heat block (heated to highest temp), place p200 tip in the wax to preheat (otherwise wax will just solidify in the cold tip)
2. Make lower master mix for “x” number of reactions, keeping all reagents cold on ice.
3. Aliquot 20μL of mix to each PCR reaction tube (use 0.5mL dome-lid thin-wall PCR tubes from Island Scientific)
4. Place lower tubes in the heat block and add 25μL paraffin wax to them – the wax will melt into the correct place
5. As soon as the wax melts, transfer them to your rack to cool (the wax will form a layer over the lower reaction mix, there will be a dimple in the middle of the wax plug but there should be no opening through to your lower reaction mix)
6. Make your upper reaction master mix, keeping all reagents cold on ice.
7. Aliquot 29μL into each reaction tube on top of the wax
8. Add your sample template to each tube (upper mix)
9. Transfer the reactions to your bench to add the control DNA (use 10 copy to measure sensitivity for first and second rounds combined and a 1000 copy control for individual rounds – if you can see a 1000 copy control after a single round, you’re doing GREAT!)
10. Start the PCR machine (MJ) and allow the block to get to 80°C before starting to load your samples

Notes:

• These primers will only work on full-length virus that has both LTRs – they will NOT work on pNL4-3

SET-UP (8-well strip tubes):

1. Place 8 well ultra-thin strip tubes (Island Scientific) into 96-well cold block. Notice the numbering of the tubes and orient correctly.
2. Make lower master mix for “x” number of reactions, keeping all reagents cold on ice.
3. Aliquot 20μL of mix to each PCR reaction tube.
4. Add one Ampliwax pellet into each tube. Place strips in the heat block unti wax melts.
5. As soon as the wax melts, transfer them to your cold block to cool (the wax will form a layer over the lower reaction mix, there will be a dimple in the middle of the wax plug but there should be no opening through to your lower reaction mix)
6. Make your upper reaction master mix, keeping all reagents cold on ice.
7. Aliquot 29μL into each reaction tube on top of the wax.
8. Add your sample template to each tube (upper mix)
9. Transfer the reactions to your bench to add the control DNA (use 10 copy to measure sensitivity for first and second rounds combined (nested) and a 1000 copy control for individual rounds – if you can see a 1000 copy control after a single round, you’re doing GREAT!)
10. Start the PCR machine (PE) and allow the block to get to 80°C before inserting your samples.

PCR Gel Purification and Cloning (Invitrogen XL TOPO PCR Cloning Kit)

Gel Purification:

1. If the PCR product is several days old, add 0.5μL of Bioline biolase and incubate for 20 min at 72°C
2. Prepare 150mL 1% agarose gel TAE buffer, microwave heat for 4 min.
3. Add 90μL of 2mg/mL of crystal violet, pour the gel and put the combs which can hold all PCR(~40μL) products
4. Add 8μL of 6X crystal loading dye to 40μL PCR product
5. Load all PCR products and 4μg HindIII run 100V for around 2 hours (note: skip one lane between each sample when you load the PCR products. It helps you to cut the gel easily without contamination)
6. Cut the right size band, put into the pre-weighted 1.5mL tube and weigh (usually i t weighs 100mg=100μL)
7. Add 250μL (2.5 volumes) of 6.6M sodium iodide and incubate at 42-50°C until the agarose is completely melted
8. Add 525μL (1.5 volumes) of binding buffer and mix well
9. Load all of the mixture onto the column, spin at 3000xg for 30 sec
10. Pour the elution in the collection vial back onto the column and repeat step 9
11. Repeat step 10 one more time to bind all the DNA to the column (a total of 3 times)
12. After the last centrifugation, discard the flow-through in the collection tube
13. Add 400μL of 1X final wash (dilute the 4x wash with ethanol) to the column and spin at 3000xg for 30 sec
14. Repeat step 13 and discard the flow-through in the collection tube after the final centrifugation
15. Spin the column again at 10,000xg for 2 min to dry the column resin
16. Transfer the column to a new 1.5mL tube
17. Add 40μL of TE buffer and incubate at room temp for 1 min
18. Spin at 10,000xg for 2 min
19. Assay 5μL by EtBr agarose gel to estimate the DNA concentration
20. Proceed directly to the TOPO Cloning reaction

TOPO Cloning and Transformation:

1. Set up the following 5μL cloning reaction per sample: | | | | —————————— | —- | | gel purified long PCR products | 4μL | | pCR-XL-TOPO vector | 1μL |
2. Mix gently and incubate for 5 min at room temp
3. Add 1μL of the 6X TOPO cloning stop solution and place on ice
4. Add 2μL of the cloning reaction into a vial of pre-thawed One Shot cells and mix gently (Do not mix by pipetting up and down)
5. Incubate on ice for 30 min
6. Heat shock the cells for 45 sec at 42°C without shaking
7. Immediately transfer the cells to ice and incubate for 2 min
8. Add 250μL of SOC medium
9. Shake the tube horizontally at 37°C for 1 hour
10. Spread 150μL from the transformation on a pre-warmed LB plate containing 50μg/mL Kanamycin
11. Incubate the plate overnight at 30°C

Plasmid mini prep (QIAgen QIAprep Spin Column Miniprep)

Note:

1. After digestion, check the insert by agarose gel. Run gel at 25V overnight
2. Once you have identified the correct clone, make a glycerol stock for long term storage. 500 μL of culture plus 75 μL of glycerol. Mix well and snap freeze in ethanol/dry ice bath.

Plasmid midi prep (Sigma GenElute HP Plsmid Midiprep)

1. Re-plate glycerol stock on LB kanamycin (50μg/mL) plate. Incubate at 30°C overnight.
2. Streak a single colony out and inoculate a 3mL culture for 8-12 hours. Add this culture to 100mL of LB kanamycin (50μg/mL) media, shake overnight at 200rpm in 30°C incubator.
3. Midi prep (see kit protocol)
4. Elute in 1.5mL of dH₂O
5. To check the insert, digest 2μL of DNA by EcoRI
6. After digestion, check the insertion size by 1% agarose gel. Run gel at 25V overnight
7. Determine the concentration by OD260 (1:50 dilution)
8. Dilute to 200 ng/μL or precipitate and resuspend to 200 ng/μL.

Sequencing:

Send 10μL of 200 ng/μL plasmid DNA combined with 7.5μL of 1μM primer in a 96 well plate. This is enough for 2.5 reactions.

1. First round of the nested PCR step of the end point dilution procedure to quantitate the cDNA.

2. First round of the nested PCR step of the limiting dilution step to amplify single templates. The conditions are exactly the same both times.

Reaction Composition

Primer positions are given for NL4-3, and yield a product ~4.6 kb. The enzyme and buffer are from Boehringer Mannheim�s Expand� High Fidelity PCR System.

Cycling Conditions

1. 94°C for 2 min, 30 sec

2. 94°C for 15 sec - 55°C for 45 sec - 68°C for 6 min for 9 cycles

3. 94°C for 15 sec - 57°C for 45 sec - 68°C for 6 min, 20 sec for 19 cycles

4. 72°C for 30 min

5. 4°C soak

Sensitivity

• Presumably one copy of pNL4-3 and one copy of cDNA.

• Provirus (8E5 cells, with one provirus per cell) down to 100 copies.

Specificity

• Nothing detected from uninfected human placental DNA.

• No apparent false priming

Protocol

1. Aliquot 150 µl of binding buffer to 50 µl of PCR product. Thoroughly mix by vigorously pipetting up and down at least 5 times (complete mixing is important).

2. Transfer mixture to 96-well filter plate.

3. Put filter plate with centrifuge alignment frame (Millipore) on top of 96-well catch plate.

4. Spin liquid through filter, into 96-well catch plate (1000x g for 5 min).

5. Discard filtrate, save catch plate for reuse.

6. Add 200 µl of 80% ethanol to each well of filter plate.

7. Spin liquid through filter, into 96-well catch plate (1000x g for 3 min).

8. Discard filtrate, save catch plate for reuse.

9. Repeat 80% ethanol wash (steps 6-8) 1 to 4 times.

10. Do one dry spin to remove residual ethanol (1000x g for 5 min).

11. Add 50 µl of 10 mM Tris-HCl pH 8.0 (preheated to 65°C) to each well. Incubate 1 minute.

12. To elute purified DNA, place the filter plate on top of a clean 96-well catch plate along with a centrifuge alignment frame and spin (1000x g for 5 min)

The PCR product is in the eluent. Transfer 10 µl to a clean catch plate with 10 µl DMSO in each well. Seal plates carefully with aluminium foil. Store both plates at -20°C.

For gels with 42 tooth combs:

M A1 B1 A2 B2 � A11 B11 A12 B12 M

M C1 D1 C2 D2 � C11 D11 C12 D12 M

M E1 F1 E2 F2 � E11 F11 E12 F12 M

M G1 H1 G2 H2 � G11 H11 G12 H12 M

For gels with 50 tooth combs:

M A1 B1 A2 B2 � A11 B11 A12 B12 M E1 F1 E2 F2 � E11 F11 E12 F12

M C1 D1 C2 D2 � C11 D11 C12 D12 M G1 H1 G2 H2 � G11 H11 G12 H12

Marker is 1 kb ladder (GIBCO-BRL).

Run approximately 2 hours (bromophenol blue marker 2/3 down lane).

Standard Mix:

Add 48 µl mix to 2µl cells in 96-well PCR plate (ISC, T-3049-1).

Cycling:

When using an Eppendorf repeat pipetter, make enough for 103 reactions and dispense 50 µl per well.

This protocol is also used for the PE 9600. Run takes 4 hours on PE 9700 and more than 4.5 hours on PE 9600.

**
**

GEL LOADING SCHEME

Check PCR products on 1% Agarose gel (2-3 µl PCR product in 15 µl total loading volume)

For gels with 42 tooth combs:

M A1 B1 A2 B2 � A11 B11 A12 B12 M

M C1 D1 C2 D2 � C11 D11 C12 D12 M

M E1 F1 E2 F2 � E11 F11 E12 F12 M

M G1 H1 G2 H2 � G11 H11 G12 H12 M

For gels with 50 tooth combs:

M A1 B1 A2 B2 � A11 B11 A12 B12 M E1 F1 E2 F2 � E11 F11 E12 F12

M C1 D1 C2 D2 � C11 D11 C12 D12 M G1 H1 G2 H2 � G11 H11 G12 H12

Marker is 1 kb ladder (GIBCO-BRL).

Run approximately 2 hours (bromophenol blue marker 2/3 down lane).

1. Fill appropriate number of 96-well plates with 110 µL LB-Amp.

2. Use 96-pin sterile replicators to replicate each original into 2 new plates, A and B.

3. Make sure rows and columns are aligned correctly (this is NOT obvious!).

4. Grow o/n (~ 20 hrs) at 37 °C in humid chamber (wet paper towels in container covered loosely).

5. Log clones that did not grow on appropiate sheet.

6. Include number of plate, date, initials.

7. Seal plates with aluminium foil.

8. Spin plates 10 min at 2000 rpm.

9. Using Transtar 96-well pipet, take off supernatant. Wash Transtar cartridge between aspirating plates as follows:
   Set up 3 pipet tip box lids; one filled with 10% bleach, two with sterile water.

   1. Pipet up/down in 10% bleach.
   2. Rinse by pipetting in first container of sterile water.
   3. Rinse again in second container of sterile water.

10. Add 100 µL LB-Amp/15% glycerol using clean Transtar apparatus.

11. Seal plates with aluminium foil.

12. Vortex briefly to resupend pellet.

13. Store at -80°C, original and plates A for Bumgarner, plates B for Katze/Mullins.

**C
**

1. Fill appropriate number of 96-well plates with 110 µL LB-Amp.

2. Thaw plates B for ~10 min.

3. Spin plates 1 min at 2000 rpm.

4. Use 96-pin sterile replicators to replicate each plate B into 1 new plate, C.

5. Grow o/n at 37°C in humid chamber.

6. Log clones that did not grow on appropriate sheet.

7. Include number of plate, date, initials.

8. Seal plates with aluminium foil.

9. Store at -80°C, plate C for Katze/Mullins

• 10X buffer

• NotI

• SalI

• ddH₂O

• Agarose

• Phenol

• Chloroform

• Ethanol

• Glycogen

• Sodium Acetate

• Linear Acrylamide

Protocol:

1. Prepare 10 µg plasmid DNA in 5 µl ddH₂O

2. Add:

a. 165 µl ddH₂O

b. 20 µl NE Buffer #3

c. 10 µl NotI

3. Vortex, spin briefly

4. Incubate overnight at 37°C

5. Run 1% agarose gel:

a. 10 µl of reaction (digested plasmid)

b. 1 µl undigested plasmid

c. 1.6 µl 1kb ladder

d. 2.0 µl phi-x174 ladder

6. Continue if plasmid was cut completely

7. Do phenol/chlorofom extraction with 100 µl P/C pH 7.5

8. Pass top layer over G25 column

9. Add:

a. 1 µl glycogen

b. 30 µl 3M Sodium Acetate pH 7.5

c. 600 µl 100% Ethanol

10. Mix, precipitate for 20 minutes at -80°C

11. Spin 12 minutes at 14,000rpm

12. Wash pellet with 70% Ethanol

13. Wash pellet with 100% Ethanol

14. Dry pellet for 5 minutes

15. Dissolve pellet in 170 µl ddH₂O

16. Add:

a. 20 µl NE Buffer for SalI

b. 10 µl SalI

17. Vortex, spin briefly

18. Incubate for 2 hours at 37°C

19. Run 1% agarose gel:

a. 6 µl of reaction (digested plasmid)

b. 1 µl undigested plasmid

c. 1 µl 1kb ladder

d. 1.4 µl phi-x174 ladder

20. Continue if plasmid was cut completely (2 bands)

21. Do phenol/chlorofom extraction with 100 µl P/C pH 7.5

22. Pass top layer over G25 column

23. Add:

a. 1 µl linear acrylamide

b. 30 µl 3M Sodium Acetate pH 7.5

c. 600 µl 100% Ethanol

24. Mix, precipitate for 20 minutes at -80°C

25. Wash pellet with 70% Ethanol

26. Wash pellet with 100% Ethanol

27. Dry pellet for 5 minutes

28. Dissolve pellet in 11 µl ddH₂O

Svetlana Mikheeva, 13 August 1999