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Physio - Pedersen Lab

Houston, Texas

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Pedersen Lab
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Lab Protocols


Luria Bertani Medium

Preparing LB Medium (Makes 1000 mL)

Reagants:

  • 1000 mL deionized water
  • 10 g Bactotryptone
  • 5 g Bacto yeast
  • 5 g NaCl several drops
  • 5 M NaOH several drops
  • 1 M HCl

Equipment:

  • 1000 mL graduated cylinder
  • stir bar
  • pH meter, with 7.0 and 10.0 pH solutions
  • 1000 mL autoclavable bottle
  • autoclave machine

Procedure:

  1. Measure about 700 mL deionized water into a graduated cylinder.
  2. Mix following into water: Bactotryptone, Bacto yeast, and NaCl.
  3. Use stir bar to mix well. While stirring, add 300 mL deionized water, making sure to wash sides of cylinder.
  4. Calibrate pH meter with 7.0 and 10.0 pH solutions. Never leave meter out of solution too long. Rinse meter with deionized water after dipping into and gently with Kimwipes.
  5. While broth mixes, adjust its pH to 7.5 with dropwise additions of NaOH and HCl.
  6. Pour broth into a 1000 mL autoclavable bottle, and autoclave on a liquid cycle.
  7. Once broth reaches room termperature, store in cold room.

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Agar Plating Solution

Preparing Agar Plates (Makes 40 Plates)

Reagents:

  • 1000 mL deionized water
  • 10 g Bactotryptone
  • 5 g Bacto yeast
  • 5 g NaCl
  • Several drops NaOH
  • Several drops HCl
  • 15 g agar
  • 1 m g 1000x ampicillin

Equipment:

  • 1000 mL graduated cylinder
  • stir bar
  • pH meter
  • 4.0, 7.0, and 10.0 pH solutions
  • 1000 mL autoclavable bottle
  • 55oC water bath
  • 40 plates
  • 37oC incubator

Procedure:

  1. Measure 700 mL deionized water in graduated cylinder. For smaller amounts, it may be more convenient to transfer water to a beaker before adding reagents.
  2. Add the following to water to make plating solution: Bactotryptone, Bacto yeast, and NaCl.
  3. Wash sides of cylinder with about 300 mL dionized water to obtain 100 mL of solution.
  4. Use stir bar to mix well.
  5. Calibrate pH meter using 7.0, 4.0, and 10.0 pH solutions. While plating solution mixes, adjust its pH to 7.5 with dropwise additions of NaOH and HCl.
  6. Add agar to large autoclavable bottle. Over agar pour pH-adjusted plating solution. Mix by swirling.
  7. Autoclave agar solution on liquid cycle.
  8. Cool in water bath for up to an hour.

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1% Agarose Gel

Preparing Solution of 1% Agarose

Reagents:

  • .9-1.0 g agarose
  • 1x TAE buffer
  • 5 m L ethidium bromide

Equipment:

  • 250 mL bottle
  • microwave

Procedure:

  1. Pour agarose into a 250 mL bottle and fill up to 100mL mark with 1x TAE buffer.
  2. Heat agarose solution in microwave for 5 minutes. Afterward, handle hot bottle with paper towel. Swirl solution to mix well. If agarose is not fully solubilized, microwave for 1 more minute.
  3. Add ethidium bromide, 5 m L / 100 mL solution, and swirl solution.
  4. Pour gel while solution remains liquid or leave at room temperature to use later.

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Mini-Prep Sol'ns 1-3

Recipes for Solutions 1, 2, and 3 of Plasmid Mini-prep

As given in Molecular Cloning Laboratory Manual, 2nd Ed. by Fritsch, Maniatis, and Sambrook

Solution 1 Reagents:

  • glucose
  • Tris.Cl (pH 8.0)
  • mM EDTA (pH 8.0)

Solution 2 Regents:

  • 0.2 N NaOH (freshly diluted from a 10 N stock)
  • SDS

Solution 3 Regents:

  • 60 mL 5 M potassium acetate
  • 11.5 mL glacial acetic acid
  • 28.5 mL H2O

Procedures:

Solution 1: Final solution should have following concentrations: 50 mM glucose, 25 mM Tris. Cl and 10 mM EDTA. Make 100 mL batch. Autoclave on 15 min wet cycle. Store at 4 oC.

Solution 2 : Final solution should be 0.2 N with respect to NaOH, and be 1% SDS. Need not be refrigerated.

Solution 3: Final solution is 3 M with respect to potassium and 5 M with respect to acetate. Need not be refrigerated.

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Polymerase Chain Rxn.

Annotations to QuickChange PCR Kit

The following steps correspond to the same numbered steps in the 1998 QuickChangeTM Site-Directed Mutagenesis Kit Handbook by Stratagene?/sup>

Setting Up the Reactions Back to Top

Step 2

This reaction corrects a mutation to give b-galactosidase activity resulting in blue colonies.
Centrifuge dry primer to settle dry powder.
Add 100 mL TE buffer to each tube, let sit 30 minutes.
For diluting primer, prepare 1:1 solution of TE:water (2x autoclaved water).
Dilute each primer to 10 pmol/mL, vortex. See example below.
Do a 1:1000 dilution of DNA template with TE. DNA template concentration should be around 1 ng/mL.
For final 50 mL volume of sample reaction, use following proportions:
1 mL dsDNA template
2 mL forward primer
2 mL reverse primer
39 mL 2x autoc. H2O

Add enzyme last. After enzyme addition briefly vortex sample. Centrifuge down.
Step 4


Allow mineral oil to drip down side of tube.

Digesting the Products

Run a Gel following digestion to check for presence of DNA

Add 10 mL DNA to 3 mL of 6x dye. Store rest of PCR on ice or freeze overnight.

Spin down enzyme.
Flick tube to mix; avoid pipetting up and down. Spin down reaction mixtures 1-2 seconds.
Transforming Supercompetent Cells

It is unnecessary to do the verification reaction using pUC18.

Add to samples 250 mL SOC broth preheated to 37 oC. Add 200 mL SOC to control.
Plate 250 mL of sample on LB/Amp plate.
Example dilution:

Stock: 37.2 nmol/100mL = .372 nmol/1mL = 372 pmol/1mL.
Do not add 1 mL primer to 362 mL buffer for proper dilution,
since margin of error for pipetting out 1 mL of liquid is too high.
Instead, add 10 mL stock primer to 362 mL of TE:water.


PMPbALf* Gauging Yield

Plasmid Mini-prep by Alkali Lysis (for Gauging Yield of DNA)

Reagents:

  • Solution 1 (50 mM glucose, 25 mM Tris.Cl [pH 8.0], 10 mM EDTA [pH 8.0])
  • Solution 2 (2 N NaOH, 1% SDS)
  • Solution 3 (5 M Potassium Acetate, glacial acetic acid, H2O)
  • Phenolchloroform
  • 24:1 Chloroform:Isoamyl Alcohol
  • 10 mg/mL RNAse
  • Isopropanol
  • 70% Ethanol
  • TE
  • 1% Agarose Gel Solution

Special Equipment:

  • Microcentrifuge
  • Microcentrifuge in cold room
  • Speed Vacuum

Procedure:

  1. Pour 1.5 mL of one bacterial culture into labeled 1.5 mL Eppendorf tube. Each Eppendorf should correspond to different culture.
  2. Centrifuge 2-3 minutes on low.
  3. Pour supernatant into large plastic beaker. Leave tubes open on bench.
  4. Aliquot 100 m L Solution 1 to each tube. Sol 1 contains EDTA, which helps break down cell walls and outer membranes.
    Vortex samples vigorously to ensure complete pellet resuspension.
    Leave on ice 5 minutes.
  5. Aliquot 200 m L Solution 2 to each sample. Solution in tubes should turn clear. SDS and NaOH aid in lysis. NaOH also denatures chromosomal DNA.
    Leave on ice 5 minutes.
  6. Aliquot 150 m L Solution 3 to each sample. K Acetate in Sol 3 binds to chromosomal DNA and helps neutralize solution.
    Mix by inverting.
    Leave on ice 5 minutes.
    Centrifuge 5 minutes on high in cold room.
    Label new set of Eppendorf tubes for supernatant.
  7. Transfer the supernatant into new tubes using the 200 mL pipetteman. Supernatant should contain only plasmid DNA.
    Under hood, add equal volume of phenolchloroform. Take from lower yellow layer. Phenolchloroform denatures proteins.
    Vortex, let sit 1 minute, vortex again.
    Centrifuge 5 minutes on high.
    Label new set of Eppendorf tubes for collecting aqueous layer.
  8. Transfer aqueous layer using 200 m L pipetteman. Dispose properly of organic bottom layer.
    Under hood add 450 m L chloroform isoamyl alcohol. Chloroform isoamyl alcohol needed to extract residual phenol, since phenol will denature RNAse, which is added later on.
    Vortex strongly, let sit 1 minute, vortex strongly again.
    Centrifuge 5 minutes on high. Label a new set of Eppendorf tubes for collecting aqueous layer.
  9. Transfer aqueous layer. Dispose properly of organic bottom layer. Last bit of aqueous layer to remain in tube appears as a bubble. Remove fluid from this "bubble".
    Add same volume of isopropanol as the amount of aqueous layer taken out.
    Mix by inverting.
    Let sit 10 minutes at room temperature.
    Centrifuge 5 minutes on high.
  10. Carefully remove isopropanol by pouring and pipetting into large beaker containing biological waste.
    Add 1 mL 70% ethanol to each tube.
    Mix by inverting.
    Centrifuge 5 minutes on high.
  11. Carefully remove supernatant by pouring and pipetting into large beaker containing biological waste.
    Speed vacuum 5 minutes. Speed vacuuming too long results in plasmid that is too hard to resuspend.
    Leave tubes open on bench.
  12. Prepare TE/RNAse solution in 1.5 mL Eppendorf tube. Add 1 part RNAse to 500 parts TE.
    Vortex TE/RNAse mixture.
  13. Add 50 m L TE/RNAse to each sample.
    Vortex each sample.
    Let sit 10 minutes at room temperature.
    Vortex each sample again and let sit for 10 minutes.
  14. Run agarose gel.

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PMPbALf* Extra Purity

Plasmid Mini-prep by Alkali Lysis (for Gauging Yield of DNA)

Reagents:

  • Solution 1 (50 mM glucose, 25 mM Tris.Cl [pH 8.0], 10 mM EDTA [pH 8.0])
  • Solution 2 (2 N NaOH, 1% SDS)
  • Solution 3 (5 M Potassium Acetate, glacial acetic acid, H2O)
  • Phenolchloroform
  • 24:1 Chloroform:Isoamyl Alcohol
  • 10 mg/mL RNAse
  • Isopropanol
  • 70% Ethanol
  • TE
  • 7.5 M Ammonium Acetate (pH 7.8)
  • 100% ethanol
  • 2 times autoclaved water
  • 1% Agarose Gel Solution

Special Equipment:

  • Microcentrifuge
  • Microcentrifuge in cold room
  • Speed Vacuum

Procedure:

Step 1.

  • Pour 1.5 mL of one bacterial culture into labeled 1.5 mL Eppendorf tube. Each Eppendorf should correspond to different culture.

Step 2.

  • Centrifuge 2-3 minutes on low.

Step 3.

  • Pour supernatant into large plastic beaker. Leave tubes open on bench.

Step 4.

  1. Aliquot 100 m L Solution 1 to each tube. Sol 1 contains EDTA, which helps break down cell walls and outer membranes.
  2. Vortex samples vigorously to ensure complete pellet resuspension.
  3. Leave on ice 5 minutes.

Step 5.

  1. Aliquot 200 m L Solution 2 to each sample. Solution in tubes should turn clear. SDS and NaOH aid in lysis. NaOH also denatures chromosomal DNA.
  2. Leave on ice 5 minutes.

Step 6.

  1. Aliquot 150 m L Solution 3 to each sample. K Acetate in Sol 3 binds to chromosomal DNA and helps neutralize solution.
  2. Mix by inverting.
  3. Leave on ice 5 minutes.
  4. Centrifuge 5 minutes on high in cold room.
  5. Label new set of Eppendorf tubes for supernatant
  6. Add .5 m L RNAse t each newly labeled tube. Ading RNAse sooner than in other plasmid prep allows phenolchloroform in Step 7B to denature the RNAses.

Step 7.

  1. Transfer the supernatant into new tubes using the 200 mL pipetteman. Supernatant should contain only plasmid DNA.
  2. Under hood, add equal volume of phenolchloroform. Take from lower yellow layer. Phenolchloroform denatures proteins.
  3. Vortex, let sit 1 minute, vortex again.
  4. Centrifuge 5 minutes on high.
  5. Label new set of Eppendorf tubes for collecting aqueous layer.

Step 8.

  1. Transfer aqueous layer using 200 m L pipetteman. Dispose properly of organic bottom layer.
  2. Under hood add 450 m L chloroform isoamyl alcohol. Chloroform isoamyl alcohol needed to extract residual phenol, since phenol will denature RNAse, which is added later on.
  3. Vortex strongly, let sit 1 minute, vortex strongly again.
  4. Centrifuge 5 minutes on high. Label a new set of Eppendorf tubes for collecting aqueous layer.

Step 9.

  1. Transfer aqueous layer. Dispose properly of organic bottom layer. Last bit of aqueous layer to remain in tube appears as a bubble. Remove fluid from this "bubble".
  2. Add same volume of isopropanol as the amount of aqueous layer taken out.
  3. Mix by inverting.
  4. Let sit 10 minutes at room temperature.
  5. Centrifuge 5 minutes on high.

Step 10.

  1. Carefully remove isopropanol by pouring and pipetting into large beaker containing biological waste.
  2. Add 1 mL 70% ethanol to each tube.
  3. Mix by inverting.
  4. Centrifuge 5 minutes on high.

Step 11.

  1. Carefully remove supernatant by pouring and pipetting into large beaker containing biological waste.
  2. Speed vacuum 5 minutes. Speed vacuuming too long results in plasmid that is too hard to resuspend.
  3. Leave tubes open on bench.

Step 12.

  1. Add 200 m L TE to each tube.
  2. Vortex each tube, let sit 10 minutes, vortex again.

Step 13.

  • Add 100 m L ammonium acetate, that is 1/2 the volume of TE.

Step 14.

  1. Add 100% ethanol, 2.5-3 times total volume in tubes.
  2. Vortex tubes, leave on ice 20 minutes.
  3. Centrifuge 10 minutes on high.

Step 15.

  1. Carefully remove supernatant.
  2. Add 500 m L 70% ethanol.
  3. Mix by inverting.
  4. Centrifuge on high 5 minutes.

Step 16.

  1. Remove supernatant.
  2. Speed vacuum 5 minutes.
  3. Resuspend in 20 m L of 2 times autoclaved water.

Step 17.

  • Run agarose gel.

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Transformation

(This protocol should be used with non-ligated plasmids)

Reagents:

  • SOC broth
  • Supercompetent cells
  • Equipment:
  • 1 15 mL conical per transformation reaction
  • Ampicillin plate(s)
  • 37 oC dry bath
  • 42 oC dry bath
  • 37 oC shaker

Procedure:

  1. Place tube of SOC in 37 oC dry bath, cool one 15 mL conical per transformation reaction on ice and thaw supercompetent cells. .
  2. Depending on DNA intensity, as seen on analytical gel, dilute plasmid 10-50 times with TE. For example, add 1 m L DNA to 49 m L TE.
  3. Add 50 m L of supercompetent cells to each pre-chilled conical.
  4. Add 0.5 m L of diluted plasmid to each conical.
  5. Lit sit 30 minutes on ice.
  6. Place ampicillin plate(s) on lab bench to warm up to room temperature.
  7. Perform heat shock: place samples in 42 oC bath for 2 minutes, and return samples to ice for 2 minutes.
  8. Vortex SOC broth and add 1 mL to each reaction.
  9. Place samples in 37 oC shaker for 1 hour.
  10. Plate 60-80 m L of each sample.

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Plating

Equipment:

  • Glass beaker containing ethanol
  • Glass stir bar
  • Petri Dish(es)
  • Bunsen burner

Procedure:

  1. Allow petri dishes to sit at room temperature for 30 minutes.
  2. Turn on Bunsen burner and pass glass stir bar over flame 3 times.
  3. Remove lid from dish, and pour or aliquot sample onto agar, making sure to hold lid face down.
  4. Flame stir bar. As soon as flame dies away, run stir bar over inside of lid to release heat.
  5. With the stir bar, spread sample evenly over agar.
  6. Flame rod twice before spreading next sample.
  7. Return to step 3 for next sample.
  8. At the end of plating allow dishes to sit right side up for 10 minutes, so that samples soak into agar.
  9. Place dishes upside down in 37 oC incubator overnight.
  10. The next day wrap dishes along their edges with parafilm, and store at 4 oC.

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Inoculation

Inoculating Bacteria

Reagents:

  • 1000x ampicillin
  • LB
  • plates with desired colonies

Equipment:

  • 1 50 mL tube
  • 15 mL tubes
  • vortex machine
  • automatic pipetter
  • 200 m L pipetteman
  • 37 oC shaker

Procedure:

  1. Take out LB broth to bring to room temperature.
  2. Thaw 1000x ampicillin, then put on ice.
  3. Label 15 mL tubes for inoculation. Each tube will correspond to a separate colony on a plate. Inoculate10 colonies per mutant following PCR and 6 colonies per mutant following ligation.
  4. Pour broth into a 50 mL tube or glass bottle. Pour as much as needed for 3 mL to go in each 15 mL tube. Run bottle top over flame before pouring.
  5. Aliquot ampicillin into 50 mL tube containing LB, about 1 mL of ampicillin per 1mL of broth. Vortex mixture.
  6. Attach measuring pipette to hand-held automatic pipetter.
  7. Run tip of pipette over flame, then fill completely with broth. Tilt broth tube to avoid contamination.
  8. Aliquot about 3 mL broth into each tube.
  9. Bring up a plate colony with pipetteman set to 80-100 m L.
  10. Empty colony into tube and mix by pipetting up and down.
  11. Place samples in 37 oC shaker overnight.

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Digestion

Plasmid Digestion, Following Mini-prep

Reagents:

  • 10x digestion enzyme buffer (1,2,3, or 4)
  • 5 m L plasmid samples following mini-prep
  • digestions enzymes
  • TE buffer

Equipment:

  • 1 1.5 mL Eppendorf tube
  • vortex machine
  • 37oC incubator

Procedure:

  1. Take out correct 10x buffer(s) to thaw. Use either buffer 1, 2, 3, or 4, depending on the digestion enzymes to be used. Once buffer thaws, leave on ice.
  2. Calculate necessary amounts of 10x buffer, plasmid, TE, and digestion enzyme(s). Keep the following in mind:
  3. 10x buffer is 1x in final solution
  4. 5 m L of each plasmid will be included
  5. Digestion enzyme volume should be kept low. Digestion enzymes are in 50% glycerol solution and final glycerol volume should be no more than 5% of total volume. For example, for a 400 m L digestion, add 40 m L of 10x buffer, 2 m L enzyme 1, 2 m L enzyme 2, and TE.
  6. Buffer should be vortexed before it is added and enzyme(s) must be added last.
  7. In Eppendorf tube combine 5 m L of each sample containing plasmid, as determined by an analytical gel.
  8. Add 10x buffer, then TE.
  9. Add digestion enzyme(s) last.
  10. Set up a 20 m L control reaction in parallel by adding the following: 2 m L 10x buffer, 2 m L plasmid, 15 m L TE, and 1 m L enzyme.
  11. Place tubes in 37oC incubator overnight (16-20 hours).
  12. Run analytical gel to check for insert. For the control, load 3-5 m L of 2x dye and 3-5 m L of plasmid. For the mutant plasmids, load 2 m L of 6x dye and 10 m L of plasmid.
  13. Keep the rest of the 400 m L digest solution(s) on ice while the gel is running.
  14. Store digest solution(s) in the ?0oC freezer.

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Precipitation Following Digestion

Reagents:

  • 3 M Na Acetate (pH 5.2)
  • 100% ethanol
  • linear polyacrilamide
  • 70% ethanol
  • TE

Equipment:

  • Cold room microcentrifuge
  • Microcentrifuge
  • Speed vacuum

Procedure:

  1. Add 40 m L (or 1/10 volume) of Na Acetate (pH 5.2) to rest of digest.
  2. Add 2.5 volumes 100% ethanol.
  3. Mix by inversion, vortex.
  4. Allow to sit at ?0 oC for 30 minutes or ?0 oCovernight.
  5. Add 5 m L polyacrilamide, vortex.
  6. Centrifuge in cold room 15 minutes on high.
  7. Remove supernatant.
  8. Wash pellet: add 500 m L 70% ethanol, vortex vigorously.
  9. Centrifuge 5 minutes.
  10. Remove supernatant.
  11. Speed vacuum 2-3 minutes.
  12. Solubilize DNA: add 20 m L TE.
  13. Vortex, let sit 15-20 minutes, vortex again.
  14. Store precipitated DNA at ?0 oC for as long as necessary.
  15. Next step is to run preparative gel in order to cut out desired DNA fragment.

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Prep. Gel and Subsqnt.Precip.

Preparative Gel and Precipitation

Reagents:

  • 1% Agarose gel
  • 1x TAE buffer
  • 6x loading dye
  • 1 kb ladder
  • TE
  • 3 M Na Acetate
  • linear polyacrilamide
  • 100% ethanol

Equipment:

  • Preparative Gel Apparatus
  • Voltage generator
  • Seran wrap
  • UV lamp
  • Autoclaved razor blade
  • 2 mL tube(s) with filter insert
  • -80 oC freezer
  • microcentrifuge

Procedure:

  1. Pour 1% agarose gel into prep gel apparatus.
  2. Prepare samples for loading: add 6 m L 6x dye to 20 m L of precipitated digested DNA sample.
  3. Load 3 m L ladder and 26 m L/sample. Load samples far apart to reduce contamination.
  4. Run the gel on 75 V. Turn off power before blue dye front nears end of gel.
  5. Place seran wrap on UV lamp and set gel (without tray) over seran wrap.
  6. Avoid gel's exposure to UV as much as possible. With UV turned on, use autoclaved razor blade to cut out DNA fragment of interest. Isolate DNA well from rest of gel to avoid contamination.
  7. Cut up fragment into smaller pieces.
  8. Using only razor blade and 2 mL tube with filter insert, scoop DNA pieces into tube.
  9. Place tube in ?0 oC freezer for ?hour, or longer if less DNA is present.
  10. Centrifuge 10 minutes at room temperature. This pools DNA and buffer to bottom of tube, leaving agarose at the top.
  11. Add 100 m L TE to agarose pellet on top.
  12. Let tube sit 10 minutes at room temperature.
  13. Place in ?0 oC freezer for ?hour.
  14. Centrifuge 10 minutes at room temperature.
  15. Discard agarose pellet and tube insert.
  16. Measure volume of DNA and add 1/10 volume of Na Acetate.
  17. Add 2-3 m L of polyacrilamide.
  18. Add 2.5-3 volume of ethanol. Follow steps 3-9 of Precipitation After Digestion.
  19. Resuspend DNA in 10-12 m L TE. Vortex, let sit 15-20 minutes, vortex.
  20. Run analytical gel to confirm presence of DNA fragment of interest. Load 1 m L/sample, along with 1 kb ladder.

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Ligation

Procedure:

1. For a 10 m L ligation reaction combine the following volumes in the order shown:

  • Expression vector 0.5 m L
  • DNA insert 4.0 m L
  • 2x autoclaved water 1.5 m L
  • 5x ligation buffer 2.0 m L
  • ATP 0.5 m L
  • DTT 0.5 m L
  • Ligase 1.0 m L

2. Incubate overnight in 16 oC water bath.

3. Store at 4 oC.

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Transformation Following Ligation

Reagents:

  • SOC broth. SOC is richer than LB and is preferable when transformation follows ligation.
  • Supercompetent cells
  • Equipment:
  • Ampicillin plate(s)
  • 37 oC dry bath
  • 42 oC dry bath
  • 37 oC shaker

Procedure:

  1. Place tube of SOC in 37 oC dry bath, cool one 1.5 mL Eppendorf per transformation reaciton on ice, and thaw supercompetent cells.
  2. Add 50 m L of supercompetent cells to each pre-chilled tube.
  3. Add 3 m L of ligation mixture to each tube.
  4. Let sit 1 hour on ice.
  5. Place ampicillin plates on lab bench to warm up to room temperature.
  6. Perform heat shock: place samples in 42 oC bath for 2 minutes, and return samples to ice for 2 minutes.
  7. Vortex SOC and add 200 m L to each reaction.
  8. Place samples in 37 oC shaker for 1 hour.
  9. Plate samples.

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Sample Prep. for Sequencing

Preparing Samples and Invoice for Sequencing Lab

1. For area of DNA sequence that needs to be verified, choose appropriate primer(s) that will provide sequencing information for that region.

2. Aliquot 2 m L of primer per sequencing reaction. Keep following in mind:

  • a. Primers must be diluted to 10pmol/m L.
  • b. Sequencing lab supplies primers M13 forward, M13 reverse, T7, T3, and sp6.
  • c. Sequence printouts generated with reverse primers show bases complementary to those on sequence map. Beginning with the reverse primer region, the map must also be read in the reverse direction.

3. Based on intensity in analytical gel, determine amount of DNA to be sent along with primer.

  • a. If there is little DNA, send 10 m L of DNA and stipulate on invoice all 10 m L should be used.
  • b. If there is relatively more DNA, send 4-5 m L.
  • c. Write down DNA concentration on invoice. Depending on gel intensity DNA concentration may vary from 150-300 ng/m L.

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Plasmid Mega-Prep

Annotations to Endo-free Plasmid Mega Protocol

The following steps correspond to the same numbered steps in the November 1998 EndoFreeTM Plasmid Mega Kit Handbook by QIAGE?/sup> .

Procedure

Step 1

  • Bring LB to room temp and inoculate 3 mL starter culture.

Step 2

  • Pour 500 mL LB into a 2 L Erlenmeyer flask. Aliquot 500 mL carbenicillin. Wrap flasks in blue diapers before putting in shaker. Pour 3 mL starter culture into Erlenmeyer flask.

Step 3

  • At this point take care of the following:
  • Pre-chill buffer P3 on ice.
  • Add RNAse to buffer P1.
  • Label centrifuge tubes and lids.
  • Transfer ?of medium to centrifuge tubes. Remove supernatant and place in large plastic beaker, then pour other half of culture into the tube for centrifugation.

Step 4

  • Use a 250 mL bottle. Make sure cartridge powder is uniformly distributed.

Step 5

  • Measure buffer in a graduated cylinder. Use a 10 mL pipette to pipette up and down and ensure complete suspension of pellet.

Step 6

  • After inverting solution looks clear.

Step 8

  • Label 1.5 mL Eppendorf tubes for samples 1-4.

Step 9

  • After turning off vacuum source, crack bottle's lid to break vacuum.

Step 10

  • After turning off vacuum source, crack bottle's lid to break vacuum.

Step 12

  • Use a 250 mL beaker to catch flow-through.

Step 14

  • Tip only holds about 50 mL, so add 4 x 50 mL of buffer QC.

Step 15

  • Use smaller centrifuge tubes. These tubes do not stand on their own, so use a styrofoam base to prop them up. Two of these smaller tubes will correspond to one sample of DNA. After eluting, swirl, and mix by transferring back and forth between two centrfuge tubes. Use pipetteman to ensure that each of the two tubes carries same volume.

Step 16

  • Add 12.5 mL isopropanol to each tube. Use usual lab stock.

Step 17

  • Add 40 mL of lab stock of 100% ethanol to kit bottle. Add 3.5 mL 70% ethanol to each tube.

Step 18

  • Leave tubes without lids on bench for as long as it takes to air dry. If there is less DNA, add only about 200 mL of endotoxin-free TE. If there is more, add about 500 mL. Place all tubes on shaker 30 min at r.t. Afterward, store tubes at 4 oC. Next day, before running gel, pool both tubes corresponding to one plasmid into 1.5 mL Eppendorf.

Preparation of Samples

After adding isopropanol and centrifuging 5 minutes, pellets are not easily visible. After removing as much supernatant as possible, add 1 mL 70% ethanol from usual stock. Centrifuge 5 minutes. Remove supernatant, speed vacuum 5 minutes. Add TE from usual stock. Vortex samples and let them sit as explained on protocol Plasmid Mini-prep by Alkali Lysis for Gauging Yield of DNA.

Agarose Gel Analysis

Load 2 m L of samples 1-4. Prepare a 1:100 dilution of DNA: add 1 m L plasmid to 99 m L usual TE stock. Also load control, i.e. plasmid from a previous mini-prep. Load as follows:

  • 1 kb ladder
  • samples 1-4
  • DNA
  • 1:100 dilution DNA
  • control.

UV Spectrophometry Analysis

Turn on UV spectrophotometer attached to terminal and let warm-up 5-10 minutes. Perform UV spec analysis on 1:500 dilutions of DNA. Into a 1.5 mL Eppendorf, add 1 m L DNA to 499 m L autoclaved water. Transfer volume to cuvette. Since water is diluant, take water absorbance reading at 260 nm. Record absorbances. Wipe away large drops of fluid in cuvette with Kimwipe between absorbance readings. See sample chart and formula under "UV Analysis" from 11/18/99. After use, cuvettes should be rinsed with water, then ethanol, and left in the proper container in the drawer on their sides.

Cleaning up

All large centrifuge tubes and lids are left to soak in water overnight. Smaller ones can be left in a beaker filled with water and larger ones are left partially filled with water and capped. The next day wash off lettering with ethanol and wash tubes and caps with soap and water. Give tubes and caps a final rinse with deionized water. Leave them to dry. Afterward, cap tubes with aluminum foil and wrap lids in foil. With the exception of the small tubes, autoclave tape is placed over the foil and everything is placed into a large plastic beaker. Cover large beaker with foil, place autoclave tape on it and autoclave on a dry cycle. It is all right if the tubes and lids are slightly wet from washing when they go into the autoclave.

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Pouring/Loading Agarose Gel

Reagents:

  • 30 mL agarose solution
  • TAE
  • 2x loading dye
  • 2 mL 1 kb ladder

Equipment:

  • analytical gel apparatus (with combs)
  • 500 m L Eppendorf tubes
  • voltage generator
  • UV lamp

Procedure:

  1. Screw the walls in on tray of analytical gel apparatus.
  2. If gel solution is in bottle in its gelatenous form, microwave 3-5 minutes until it turns liquid.
  3. Aliquot 150 mL of liquefied gel solution along two edges of tray where walls were screwed in, to ensure tray is "sealed".
  4. Insert combs then pour about 30 mL agarose solution up to the edge of tray's low-lying walls.
  5. Leave tray alone for 30 minutes while gel turns to gelatenous solid.
  6. Wet the surface of gel with TAE and slowly remove combs.
  7. Lower walls of tray and place tray in gel apparatus such that DNA migrates toward positive end.
  8. Pour enough TAE into apparatus to fill large side wells and cover surface of gel.
  9. Prepare samples in 500 mL Eppendorf tubes. Add 1 mL of 2x loading dye to each tube.
  10. Vortex DNA samples if they have been sitting in fridge. Add 1 mL of sample to each tube containing dye. Mix by pipetting sample up and down and leave pipette tip in tube to use for loading.
  11. Load 2 mL of each of the following into separate wells: 1 kb ladder, control, samples. Do not use gels older than 3 days.
  12. Cover apparatus and connect to voltage generator.
  13. Turn voltage generator to 75 V and let gel run for about 25 minutes. Gel runs more slowly with fewer samples and/or lower voltage. For larger pieces of DNA like plasmids, let gel run until blue color front nearly reaches end of gel. For smaller pieces of DNA do not let blue front come too close to the end.
  14. Turn off power supply and remove tray.
  15. Remove gel from tray and view under UV light to see DNA. DNA may be hard to see if gel is 2 or 3 days old.
  16. If DNA is not clearly visible under UV, place gel in a container, such as a small plastic tray, and add water to container until gel surface is covered. Add 2 mL of ethidium bromide to water and place container on shaker for 10 minutes. Again view gel under UV light.
  17. Take picture of gel. If using a camera cabinet, center gel in cabinet. Close cabinet door, and switch on UV light. Open "image" program on computer desktop. Focus image of gel using focus knobs on cabinet, and by clicking "exposure". Click "print" to take picture. Switch off UV light, and remove gel from cabinet. Wipe glass surface in cabinet with Kimwipes.

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Examine dTc Binding

Preparation of Solutions and Cells

BSA Ringer (ph 7.0)

  • 30 mL Ringers sol.
  • 30 uL BSA - mix = 0.1% BSA sol.
  • keep on ice

1:10 Dilution of Carb Stock

  • 900 uL of 0.1% BSA sol.
  • 100 uL carb. (0.1 M carb. stock in H2O)
  • keep on ice

10 mM dTC/water solution

  • make serial dilutions (see chart)

1000 uL of 0.56 nM High Activity 125I Bgtx Stock Sol.

  • 1000 uL 0.1% BSA Ringers
  • 5.6 uL Bgtx

0.4 M Bgtx (cold)

  • 5 mL BSA sol.
  • 8 uL Bgtx stock (vial)
  • more then exact amount needed

Cell Prep Procedure

  • use 2 pitri dishes of cells; pour off media of one plate
  • scrape cells from surface of the pitri dish with media; pour mixture into second dish; rescrape
  • using 15 mL centrifuge tube, pipette mixture (media cells) inot this tube - rince dish with K Ringer sol. + scrape again
  • combine with mixture in centrifuge tube
  • bring up to 15 mL with 0.1% BSA sol.
  • centrifuge 2 mins. @ 1800 RPM
  • wash + resuspend in 15 mL 0.1% K Ringer
  • centrifuge 2nd time 2 mins. @ 1800 RPM
  • pour off wash
  • to the cells add 1800 uL of 0.1% BSA/K Ringer sol.
  • vortex + use in step 3 of experiment proper; bring vol. up to 3 mL with BSA sol.
  • note: keep on ice
  • note: vortex at each pipetting to keep cells in suspension

Experiment Proper

Materials

  • 12x75 mm PP tubes (2 sets) + extras
  • 2 test tube racks
  • 10 eppendorf tubes (label A - J)
  • ice + ice bucket
  • 500 mL beaker (waste)
  • 500 mL beaker (pipette displosal prior to Bgtx)
  • cells on pitri dishes (above)
  • filters: GFL or GFC
  • filtering device
  • radioactive waste bay + tape + stickers

Protocol I

  • make up solutions except hot/cold Bgtx sols.

Protocol II

  • label eppendorf tubes/caps A through J
  • tube A: add 12 uL of dTc (ligand) to 288 uL BSA
  • tubes B, D, F, H, J: add 210 uL BSA sol.
  • tubes C, E, G, I: add 180 uL BSA sol.
  • pipette 90 uL from tube A; dispense + mix in tube B; continue through J
  • add 20 uL to binding assay chart labaled dTc binding; hold until called for
  • serial dilutions for ligands are done in eppendorfs
  • according to conc. of final solution, vol. of ligands can be calculated
  • 0.1% BSA Ringers sols. added into eppendorfs first, then corresponding volumes of ligands are added
  • Beware: vortex the sol. at each dilution
  • directions for usage in assay preparation will be given at that step

Protocol III

  • see chart (below) on dTc binding:
tubes 1-3 add 20 uL 0.1% BSA Ringer sol.
tubes 37-39 add 20 uL 0.1% BSA Ringer sol.
tubes 34-36 add 14 uL 0.1% BSA Ringer sol. + 6 uL 1:10 dilution of BSA 0.1% carb.
  • incubate 30 mins. on shaker

Protocol IV

  • see chart (below) on dTc binding:
tubes 1-3 add 0 vol. dTc dilution
tubes 4-6 add 20 uL dTc dilution vial J (eppendorf tubes)
tubes 7-9 add 20 uL dTc dilution vial I (eppendorf tubes)
  • continue through 31-33; should use vials
tubes 34-36 no dTc added
tubes 37-39 no dTc added
  • be sure to vortex at each pipetting
  • tubes 1-3, 37-39 are controls
  • since going from lowest dilution to highest, no need to change tips

Protocol V

  • to each tube 1-3, 4-6, etc. through 37-39 add 40 uL/tube of cell suspension
  • vortex after each pipetting; keep cells in suspension
  • incubate 30 mins. on shaker; cover + parafilm

Protocol VI

  • prepare 125I Bgtx solution

    1000 uL of 0.56 mM high sctivity (209) Bgtx 125I
    1000 uL BSA sol. add 5.6 uL Bgtx 125I

    • to be done under fume hood
    • displose of pipettes, gloves, tubes, and tips in radiation waste bag
    • area of work desk should be taped in radioactive tape, tubes should be labeled with rad. stickers
    • identify date and amt. used of Bgtx on stock chart
    • to each tube after 30 mins. incubation/shaking, add 20 uL of hot Bgtx 125I; vortex at each pipetting
    • incubate 2 hrs. (with cover); place on shaker
    • add Bgtx every 20 secs.; record your time (set on time up)
    • prepare filters: soak in 1% PEI sol. for 2 hrs. at room temp.
    • set up filtering aparatus
    • change filtering aparatus and make sure it works properly
    • vacuum should be within 5 mm Hg (valve setting)
    • rinse 2 or 3 times (cold)
    • when ready for use: filters are placed on apparatus - rinse with PBS 2 or 3 times (hot)
    • vacuum
    • have numbered tubes 1-39 + 5 extra tubes
    • remove filters; have tubes pre labeled 1-39, remove filters + place in bottom of corresponding tube

    Protocol VII

    • after 2 hs. incubation of hot Bgtx I125 in presence of cell ligand:
    • add cold Bgtx sol. 0.4 uM (80 uL/tube)
    • use dTc binding chart...count and add every 20 secs.
    • keep on ice
    • waste - tubes A, B, C...cold Bgtx
    • waste - measure filters e.g. 600 mL, to 2 tubes add 500 uL
    • see filtration method via chimney filtering apparatus

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