Glossary of Protocols

Making Competent Cells:

1. Plate DH5 alpha seed stock and grow overnight at 37C
2. Isolate colony from plate into LB broth tube and grow overnight
3. Inoculate 12.5 mL of SOB media with 150 uL of fresh overnight culture
4. Incubate SOB culture at 37C in shaking incubator for 2 hrs
5. Aliquot into 12 eppi tubes (1 mL per tube)
6. Put on ice for 10 minutes
7. Spin down for 10 minutes at 4000 rpm
8. Poor off all excess liquid (carefully pipette it out if needed)
9. Re-suspend pellet in 333 uL of TB per tube
10. Incubate on ice for 10 minutes
11. Spin down for 10 minutes at 4000 rpm
12. Poor off all excess liquid
13. Re-suspend pellet in 83 uL of TB per tube
14. A 5.83 uL of DMSO to each tube
15. Incubate on ice for 10 minutes

Notes:
• Prepare empty tubes by freezing at -20C before use
• Keep cells on ice as much as possible
• Divide into the required aliquots of 50 or 25 uL
• Store finished cells in -80C freezer

Gel Electrophoresis

1. Measure 0.5 g agarose powder and add it to a flask
2. Add 50 mL of TAE buffer to flask
3. Microwave flask or incubate in hot water bath until agarose solution has become clear
4. Let the solution cool to about 50-55C occasionally swirling to mix
5. Add 2-5 uL of Ethidium Bromide to agarose solution and swirl to mix
6. Secure the rails on either side of the gel tray in the up position and carefully pour agarose solution into casting tray.
7. Place combs in casting tray
8. Allow solution to cool and solidify
9. Loosen and lower rails on either side of the casting tray
10. Gently place tray in gel box.
11. Add TAE buffer until it is just over the surface of the gel
12. Carefully pull out the comb

Notes:

• To prepare samples, add 1 uL of Loading dye per 5 uL of sample
• Add 10-20 uL of sample to a given well
• Gel box is turned to 135 V and run until the darkest band reaches the center of the gel.

LB Agar plates

1. Add the following to an empty 1000 mL flask
2. 7 g of tryptone
3. 3.5 g yeast extract
4. 3.5 g NaCl
5. 10.5 g agar
6. Add Distilled H2O to final volume of 700 mL
7. Autoclave solution for 30-45 minutes on liquid cycle
8. Allow flask to cool in water bath
9. Add antibiotics when flask roughly 50-55C
10. Mix well by swirling
11. Label plates to be poured with media, antibiotic, and date poured
12. Pour plates, filling each with media until ~1/2-2/3 full flaming the mouth of the flask between each plate
13. Allow plates to cool at room temperature overnight
14. Put plates back into sleave, seal the sleave, and label it
15. Store plates in refrigerator

LB broth tubes

1. Add the following to a 1000 mL flask
2. 10 g tryptone
3. 5 g yeast extract
4. 5 g NaCl
5. Add distilled H2O to final volume of 1000 mL
6. Adjust to final pH of 7.0 with NaOH
7. Distribute into tubes (5 mL per tube) or bottles
8. Autoclave for 30-45 minutes on liquid cycle

SOB Media

1. Add the following to a 1000 mL flask or beaker
• 20 g tryptone
• 5 g yeast extract
• 0.6 g NaCl
• 0.2 g KCl
2. Add distilled H2O to final volume of 1000 mL
3. Autoclave for 35-40 minutes on liquid cycle
4. Add the following sterile reagents
   1. 10 mL of 1M MgSO4
   2. 10 mL of 1M MgCl2

Notes: • It is recommended that SOB be made in smaller batches to prevent contamination of a large amount of it SOC Media 1. To 1000 mL of SOB add the following: 2. 20 mL of 1M sterile glucose Notes: • See notes on SOB media Thioglycolate Media Reinforced Clostridial Media To a 1000 mL flask add the following: 1. 10 g beef extract 2. 10 g peptone 3. 5 g NaCl 4. 5 g dextrose 5. 3 g yeast extract 6. 3 g sodium acetate 7. 1 g soluble starch 8. 0.5 g L-Cysteine HCl 9. 0.5 g agar 10. Adjust pH to 6.8+/-.2 at 25C Addition of resazurin is optional. This media was prepared anaerobically with nitrogen gas in an anaerobic hood. Antibiotics Table 1000x ampicillin 10mg/mL in distilled water 1000x chloramphenicol 34mg/mL in 100% ethanol 1000x X-gal 20mg/mL in DMSO Antibiotic Spread Plates 1. Dilute the antibiotic of interest (25 uL of antibiotic in 75 uL of appropriate solvent) 2. Pipette 100 uL of the solution onto the each plate and spread with beads 3. The plates must be allowed to dry and the antibiotic soak in (this usually takes at least an hour) Phosphotase Restriction Digest Ligation Chemical Transformation Plasmid MiniPrep The following protocol uses reagants from the IBM plasmid mini-prep kit 1. Spin down LB broth cultures 1.5 mL at a time until most or all of the tube is gone. a. Each LB tube will require at least one eppi tube 2. For each repetition of the previous step poor off the supernatant 3. Re-suspend the pellet in 200 uL of PD1 buffer 4. Add 200 uL of PD2 buffer and mix by inverting 10 times 5. Wait 2 minutes 6. Add 300 mL of PD3 buffer. Mix by inverting 10 times 7. Centrifuge for 3 minutes at 16xg 8. Place PD column into a 2 mL collection tube 9. Add supernatant from step 7 to the PD column and centrifuge at 16xg for 30 seconds 10. Discard flow-through and place the PD column back in the 2 mL collection tube 11. Add 400 uL of W1 buffer to PD column 12. Centrifuge at 16xg for 30 seconds 13. Discard flow-through and place PD column back in collection tube 14. Add 600 uL of Wash buffer to PD column 15. Centrifuge at 16xg for 30 seconds 16. Discard flow through and place PD column back in collection tube 17. Centrifuge again to dry at 16xg for 3 minutes 18. Transfer PD column to 1.5 mL eppi tube 19. Add 50 uL of MilliQ H2O for elution to the center of the PD column 20. Let stand for 2 minutes 21. Centrifuge at 16xg for 2 minutes 22. Discard PD column, close and label eppi tube. 23. Nano drop to determine concentration Genomic MiniPrep PCR The following protocol requires bulls eye Taq Polymerase Master Mix. For each pcr reaction add to a single pcr tube the following reagents: 1. 12.5 uL Taq Master Mix (final concentration: 1x) 2. Forward Primer (final concentration: 0.1-1.0 uM) 3. Reverse Primer (final concentration: 0.1-1.0 uM) 4. Distilled or MilliQ water (to total final reaction volume of 25 uL) 5. Template DNA (4 uL of 1:50 diluted LB culture) 6. Total Volume of 25 uL Once the previous reagents have been mixed do the following: 1. Mix reaction gently by pipetting the solution up and down a few times 2. Run the PCR reaction using the following thermocycler program a. 95C for 5 minutes b. 95C for 30 seconds c. 68C for 30 seconds d. 72C for 45 seconds e. 72C for 5 minutes 3. Steps b, c, and d should be run 20-30 times Gel Extraction TOPO TA cloning