Transformations (Electroporation or Heat Shock)

To thaw:

1. Competent cells (on ice) ... Found in -80 freezer
2. SOC medium (@ room temp.) ... Found in -80 freezer
3. Turn on shaking incubator
4. Turn on water bath (Heat shock only)
5. Agar plates in incubator
6. The DNA (@ room temp)

Electroporation

1. Combined 40 μL of electrically competent DH5a cells and 1 μL of ligated DNA to an Eppendorf tube.
2. Transferred the contents of the Eppendorf tube to a cuvette and lightly tapped the cuvette on the table to evenly distribute the contents and to get rid of air bubbles.
3. Placed the cuvette into the Bio-Rad MicroPulser and delivered the electric shock.
4. Immediately after, added 900 μL SOC medium to the cuvette and micropipette mixed the solution.
5. Transferred the solution from the cuvette to a shaker tube and placed in the shaker at 37°C at 200 rpm for 1 hour.
6. After shaking for 1 hour, streaked 150 μL of the solution onto an agar plate with the respective antibiotics.
7. Incubated plates at 37°C for at least 24 hours.

Heat Shock

1. Thawed One Shot TOP10 chemically competent cells on ice.
2. Added 2 μL of DNA sample into competent cells
3. Incubated the cells on ice for 35 minutes.
4. After the ice incubation, placed the samples into a 42° C water bath for 30 seconds.
5. Quickly took them out and immediately added 250μL of SOC medium
6. Placed the samples into a 37° C shaking water incubator for 1 hour at 200 rpm.
7. After shaking for 1 hour, streaked 150 μL of the solution onto an agar plate with the respective antibiotics.
8. Incubated plates at 37°C for at least 24 hours.

Recipe for 1 liter of LB media AGAR PLATES

1. Add the following in a 2000 mL E. flask and stir it a bit:
   • 10g Tryptone
   • 5g Yeast Extract
   • 10g NaCl
   • 1 liter of water
   • Add 15g agar powder
2. Autoclave at 45 minutes (while it is being autoclaved, prepare the incubator bath at 55 degrees Celsius and label plates)
3. After autoclave, place flask in 55 degrees celsius incubator
4. After the medium has cooled down a bit (but not too cool or else the medium will solidify), add _____ antibiotics amount based on plasmid you are working with and stir flask
   • Ex: 1 mL of ampicillin if working with DinoIII-GUS plasmid
5. Pour plates near bunsen burner

Recipe for 1 liter of LB media SOLUTION

6. Add the following in a 1000 mL E. flask and stir it a bit:
   • 10g Tryptone
   • 5g Yeast Extract
   • 10g NaCl
   • 1 liter of water
7. Autoclave at 45 minutes
8. After autoclave, place flask in 55 degrees celsius incubator
9. After the medium has cooled down a bit (but not too cool or else the medium will solidify), add _____ antibiotics amount based on plasmid you are working with and stir flask

Protein Expression/Isolation Protocol

Protein Expression

Initial 10 mL Overnight Culture
1. Added 10 mL of LB and 10 μL of Ampicillin to a 50 mL falcon tube
2. Used a p10 tip to scrape the ice off of the glycerol stock and ejected the tip into the falcon tube.
3. Incubated for 18 hours at 37° and 200 rpm.

500 mL Mass Culture
1. Dumped the grown up 10 mL overnight culture from the day before into 500 mL of LB + 500 μL Ampicillin and incubated at 37°C for 3-6 hours at 150 rpm.
   • Incubation time will depend on absorbance at OD 600, check periodically while the culture incubates, needs to be between 0.6 and 1.0.
2. Once the absorbance was in that range, added 500 μL of 1M IPTG to the 500 mL culture and incubated at 30°C and 150 rpm for 18 hours.

Pelleting Cells and Resin Binding

3. Evenly divided the 500 mL mass culture into 10 different falcon tubes and centrifuged at 5,000 rpm for 15 minutes to pellet the cells.
4. Discarded the supernatant from each of the tubes and resuspended the cells in 25 mL of 1X PBS with 1 mM DTT.
   • Prepared PBS from a 10X stock solution: 5 mL of 10X PBS and 45 mL of diH2O
   • Prepared 1M DTT by dissolving 0.15424 grams of DTT in 1 mL of diH2O
   • Then combined 25 mL of 1X PBS and 25 μL of 1M DTT, mixed, and used as resuspension buffer above.
5. Once all of the pelleted cells were fully dissolved in 25 mL of 1X PBS with 1 mM DTT,added a the solution and fully dissolved. To help withProtease Inhibitor Cocktail Tablet to this, vortexed repeatedly.
6. Once fully dissolved, added 2 volumes of a scoopula tip full of Sigma CellLytic Express powder to the mixture and vortexed.
7. Allowed the solution to sit benchside for 1 hour..
8. Sonicated the solution with short pulses while on ice.
9. Centrifuged the lysed cell solution at 14,000 rpm for 15 minutes (keep everything, supernatant and pellet are used in step 11).
10. While that ran, centrifuged 1 mL of Pierce Gluthathione Agarose at 13,000 rpm for 1 minute and removed the ethanol.
    • Washed the resin (pellet) with 1X PBS with 1 mM DTT, spin down at 13,000 rpm for 1 minute, and removed the supernatant. Repeated this step 3 times
11. Then once the 1X PBS with 1 mM DTT is removed from the final wash step, resuspended the resin in the supernatant that comes from the lysed cells centrifugation from step 9.
    • Made sure to keep some of the cell debris from step 9 to run in the SDS gel and use in the Western blot
12. Shook at 215 rpm at 4°C overnight (shaker in fridge?).

Protein Isolation

13. Continuing from the day before, prepared a column by running diH2O through it.
14. Poured the chilled solution containing the resin through the column and collected the flow through. Labelled as flow through.
15. Poured 3 mL 1X PBS with 1 mM DTT through the spin column as a wash step and collected the flow through. Labelled as Wash 1.
16. Repeated step 15 twice more for a total of 3 washes.
17. Measured and recorded the absorbance at 280 using a UVette for each wash step.
18. Prepared a 10 mM solution of Glutathione (reduced) by dissolving 0.0307 grams in 10 mL of 1X PBS with 1 mM DTT. This will be used for elution.
19. Poured 3 mL of the elution buffer from step 18 through the column and collected the flow through. Labelled as Elution 1.
20. Repeated step 19 twice more for a total of 3 elutions.
21. Measured and recorded the absorbance at 280 using a UVette for each elution step.

Ligation

1. Added 6 μL of diH2O to a clean 1.5 mL Eppendorf tube, 1μL of T4 DNA Ligase Buffer, 1μL of plasmid DNA and 1μL of DNA part and mixed.
2. Added 1 uL of T4 DNA ligase
3. Pipet mixed the tube and incubated at room temperature for 10 minutes.

QIAGEN Plasmid Midi Kit Protocol

Did you do your glycerol stocks?
1. Separated 500 mL of bacterial overnight culture into 2 sets of 5 separated 50 mL falcon tubes and centrifuged at 5,000 rpm for 15 minutes at 4°C.
2. Decanted supernatant.
3. Added 10 mL of Buffer P1 to two tubes, pipet mixed, and transferred to another tube.Mixed and transferred contents to the next tube with pelleted cells. Repeated until all tubes were combined.

4. Added 10 mL of Buffer P2 to the tube containing 10 mL of Buffer P1 and the combined resuspended ?pelleted cells and vigorously inverted 6 times.
5. Incubated at room temperat?ure for 3 minutes.
6. Added 10 mL of Buffer P3 (Buffer P3 and N3 is the same) and vigorously inverted 10 times.
7. Incubated on ice for 15 minutes.
8. Centrifuged at 5,000 rpm at 4°C for 30 minutes.
9. Once centrifuged, transfer clear supernatant to 2 high speed centrifuge tubes while avoiding all of the flakes on the sides and in the solution.
10. Centrifuged the tube again at 14,000 rpm at 4°C for 15 minutes
11. While this ran, equilibrated the QIAGEN-tip by adding 4 mL of QBT to the QIAGEN-tip.
12. Added the clear solution (from step 10) to the QIAGEN-tip and allowed it to enter the resin by gravity flow
13. Next, add 10 mL of Buffer QC to the QIAGEN-tip and allow gravity to drip.
14. Once that passed through, added 10 mL more of Buffer QC and allowed it to flow through.
15. Add 5ml buffer QF into the column to elute the DNA into a clean high speed plastic tubes. (USE GRAVITY FLOW)
16. Precipitate DNA by adding 3.5ml room-temperature Isopropanol to the eluted DNA and mix. Centrifuge at 14,000 rpm for 30min at 4C.
17. Carefully removed the supernatant making sure not to disrupt the clear pellet.)
18. Added 2 mL of room-temperature 70% ethanol and centrifuged for 10 minutes at 14,000 rpm at 4°C. Discarded the supernatant leaving as little liquid behind as possible, careful -not to disrupt the clear pellet.
19. Air-dried the pellet for 20 minutes in the vent hood and redissolved in 100 μL of Buffer EB.

Overnight Cultures

To Thaw:

1. Antibiotics (Thaw @ room temp.) ... Found in freezer
   • Added about 5-7 mL of LB to a 25 mL Falcon tube along with 5-7 μL of antibiotics (Chloramphenicol for pSB1C3; Kanamycin for pCB302; Ampicillin for DinoIII)
   • Dipped a p10 tip into your selected colony and dropped into the tube
     • If using a glycerol stock, simply scrapped some of the ice with the p10 tip and dropped into the tube
       • NOTE: did NOT let the glycerol stock defrost!
     • If you want to do overnight colony PCR from the same colonies:
       • Used the tip of a pipette or toothpick to pick a colony then swirled it around in 10 μL of DI water
       • Added 1 μL into PCR tube and another μL into overnight cultures
   • Incubated at 37° C at 220 rpm for 16-18 hours

PCR Protocol

To thaw:

1. Mastermix (on ice) ... Found in freezer
2. Primers (on ice) ... Found in freezer
3. 20 μL Reaction
   • Prepared a PCR concentration cocktail with the following proportions: 7 μL of diH2O,10 μL PCR Mastermix, 1 μL of the forward primer, and 1 μL of the reverse primer.
   • Added 19 μL of the concentration cocktail into a PCR tube along with 1 μL of the DNA.
   • Placed PCR tube in the thermocycler at the following generic settings:
     • 95° C for 3:00 minutes
     • 95° C for 1:00 minute
     • 52° C for 1:00 minute *Annealing temperature varies depending on primer
     • 72° C for 1:00 minute
     • 30X (Go to Step 2)
     • 72° C for 5:00 minutes Lid Temperature: 105° C
4. Turn on water bath (Heat shock only)
5. Agar plates in incubator
6. The DNA (@ room temp)

Colony PCR Protocol

1. 20 μL Reaction
   • Prepared a PCR concentration cocktail with the following proportions: 7 μL of diH2O,10 μL PCR Mastermix, 1 μL of the forward primer, and 1 μL of the reverse primer.
   • Added 19 μL of the concentration cocktail into a PCR tube along with 1 μL of the DNA.
   • Using a 10 μL micropipette, touched the tip onto the selected colony and swirled around in the PCR tube.
   • Placed PCR tube in the thermocycler at the following generic settings:
     • 95° C for 3:00 minutes
     • 95° C for 1:00 minute
     • 52° C for 1:00 minute *Annealing temperature varies depending on primer
     • 72° C for 1:00 minute
     • 30X (Go to Step 2)
     • 72° C for 5:00 minutes Lid Temperature: 105° C
2. Turn on water bath (Heat shock only)
3. Agar plates in incubator
4. The DNA (@ room temp)

Regular Restriction Digest Protocol

To thaw:

1. Restriction enzyme (on ice) .... Found in freezer
2. Fast Digest buffer (on ice) ... Found in freezer
3. Turn on water bath
4. 30 μL Fast Digest Restriction Digest
   • Prepared a Fast Digest concentration cocktail with the following proportions: 1 μL Restriction Enzyme #1, 1 μL Restriction Enzyme #2, 3 μL of 10X Fast Digest Buffer,and 15 μL of diH2O.
   • Added 20 μL of this cocktail to a clean 1.5 Eppendorf tube and then added 10 μL of DNA
   • Incubated at 37° C for 30 minutes.

Purpose: To digest/linearize large amounts of plasmid for gel extraction

Large Restriction Digest Protocol

5. 250 μL Fast Digest Restriction Digest
   • Prepared a Fast Digest concentration cocktail with the following proportions: 25 μL Restriction Enzyme #1, 25 μL of 10X Fast Digest Buffer, and 100 μL of diH2O.
   • Incubated at 37° C for 3 hours.

Purpose: To inactivate the restriction enzymes before a ligation is performed

6. Heat Kill

   • Placed digested DNA in water bath at 65° C for 20 minutes