Difference between revisions of "Team:CSU Fort Collins/Protocols"

PCR

Materials

• Template DNA
• 5X HF Phusion Buffer or 10X HF Taq Buffer
• Molecular Grade Water
• dNTPs
• Primers
• DNA Polymerase (Phusion or Taq)

Procedure

Piece Amplification

1. Create the following mixture:

Component	50μl reaction
Molecular grade H2O	Added first. 32.5μL
5x HF Buffer	10μL
10 mM dNTPs	1.0μL
Primer A (10μM)	2.5μL
Primer B (10μM)	2.5μL
Template DNA	1.0μL
DNA polymerase	Added last. 0.5μL

2. Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:

Cycle Step	Temp(C)	Time	Cycles
Initial denaturation	98	5 min	1
Denaturation	98	10s	35
Annealing	LowerTm+3	15s	35
Extension	72	45s/kb	35
Final Extension	72	10min	1
Hold	4	Hold	1

Colony PCR

1. Create the following mixture:

Component	50μl reaction
Molecular grade H2O	Added first 33.5ul
5x Phusion HF Buffer	10μL
10 mM dNTPs	1.0μL
Primer A (10μM)	2.5μL
Primer B (10μM)	2.5μL
Colony	1
Phusion DNA polymerase	Added last. 0.5μL

3. Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:

Cycle Step	Temp(C)	Time	Cycles
Initial denaturation	98	5 min	1
Denaturation	98	10s	35
Annealing	LowerTm+3	15s	35
Extension	72	45s/kb	35
Final Extension	72	10min	1
Hold	4	Hold	1

PCR with Q5

Materials

• Template DNA
• Q5 master mix
• Molecular Grade Water
• Primers

Component	50μl reaction
Molecular grade H2O	Added first. 32.5μL
Q5 Master Mix	25μL
Primer A (10μM)	2.5μL
Primer B (10μM)	2.5μL
Template DNA	1.0μL

Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:

Cycle Step	Temp(C)	Time	Cycles
Initial denaturation	98	5 min	1
Denaturation	98	10s	35
Annealing	LowerTm+3	15s	35
Extension	72	30s/kb	35
Final Extension	72	10min	1
Hold	4	Hold	1

Colony PCR

1. Create the following mixture:

Component	50μl reaction
Molecular grade H2O	Added first 32.5ul
Q5 Master Mix	25μL
Primer A (10μM)	2.5μL
Primer B (10μM)	2.5μL
Colony	1

2. Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:

Cycle Step	Temp(C)	Time	Cycles
Initial denaturation	98	5 min	1
Denaturation	98	10s	35
Annealing	LowerTm+3	15s	35
Extension	72	30s/kb	35
Final Extension	72	10min	1
Hold	4	Hold	1

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Digestion

Materials

• 2 LB + Antibiotic plates
• Spreaders or glass beads
• LB Media
• Ligated back bone
• Digested back bone(control)

Procedure

1. Create the following mixture:

Component	50μl reaction
Molecular Grade Water	Calculated. Use to make reaction total volume = 50ul
Custsmart Buffer(different if using pstI)	5ul
DNA	1ug
Enzyme 1	.5ul
Enzyme 2	0.5ul

2. Digest your mixture in a water bath at 37C for 60min

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Ligation

Ligation Using the “Ligation Template” excel sheet, calculate the amount of each component to combine in a ligation mix for an Insert: Backbone ratio of 4:1

Component	20μl reaction
Molecular Grade Water	Calculated. Use to make reaction total volume = 20ul
T4 DNA Ligase Buffer	2ul
Insert DNA
Backbone DNA
T4 DNA Ligase	1ul

Incubate at 16C overnight on a heating block or at room temperature for 1 hour.

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Transformation

Materials

• 2 LB + Antibiotic plates
• Spreaders or glass beads
• LB Media
• Ligated back bone
• Digested back bone(control)

Procedure

1. Set water bath to 42C
2. Remove LB+Antibiotic plates from 4C and allow them to come to RT.
3. Thaw chemically competent cells on ice. Leave in microcentrifuge tube.
4. Add 5μL of ligation mix chemically competent cells.
5. Add 5uL of digested back bone to chemical competent cells as a control
6. Incubate on ice for 30 min.
7. Heat shock cells for 60 sec at 42C without shaking.
8. Aseptically (by the fire or in the hood) add 250μL of LB media to the tube (DO NOT ADD ANTIBIOTIC AT THIS STEP). Cap tightly.
9. Place tube horizontally in shaker. Incubate at 37oC and 225 rpm for 1 hr.
10. In the laminar hood, spread 100uL of transformants onto LB+Antibiotic plates.
11. Leave plates in 37C incubator overnight. Store remaining liquid cultures in 4oC.

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LB Media

Materials

• LB Miller (Powder)
• 1L Glass Bottle
• Stir Bar
• DI/RO Water

Procedure

1. Triple rinse bottle with DI water
2. Add stir bar to the bottle
3. Add 700 mL of DI Water to the 1L Bottle
4. Add 17.5 g of LB Miller
5. Put of stir plate and stir until large clumps are dissolved (Remaining small clumps will dissolve in Autoclave)
6. Tighten the cap all the way and loosen with 2 full turns to the left [IMPORTANT for SAFETY]
7. Place autoclave tape on top
8. Autoclave on Liq 30
9. Let cool and then tighten the cap and store at room temperature

LB Agar Media for Plates

Materials

• LB Miller (Powder)
• 1L Glass Bottle
• Stir Bar
• DI/RO Water
• Agar
• Antibiotic if applicable
• Petri Dish Plates
• Ethanol Proof Markers
• Tape

Procedure

1. Triple rinse bottle with DI water
2. Add stir bar to the bottle
3. Add 700 mL of DI Water to the 1L Bottle
4. Add 17.5 g of LB Miller
5. Add 8.4g of agar (not agarose)
6. Put of stir plate and stir until large clumps are dissolved (Remaining small clumps will dissolve in Autoclave)
7. Tighten the cap all the way and loosen with 2 full turns to the left [IMPORTANT for SAFETY]
8. Place autoclave tape on top
9. Autoclave on Liq 30
10. Remove IMMEDIATELY from autoclave when finished
11. Place on stir plate and slowly stir (no air bubbles from stirring too fast) until the bottle can be held comfortable for a 7 seconds (20-30 min of cool down time)
12. Add 700 μL of aliquoted Antibiotic
13. INSIDE THE HOOD:
    1. Spray the sleeve of plates, the agar media, the marker, and the tape down with ethanol before putting them in the hood. When opening the sleeve of plates, be careful to not rip it because you will put the plates back into it.
    2. Following sterile technique pour the plates about a 3rd of the way full
    3. Allow to cool in the hood until they have solidified and are no longer warm
    4. Turn the plates upside down and put back in the sleeve
    5. Tape the sleeve shut and write the antibiotic and the date they were made on the tape.

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Gel Electrophoresis

Materials

• Agarose
• 1X TAE Buffer
• Parafilm
• Electrophoresis chamber and power source
• SYBR Green
• Loading Dye
• DNA Ladder
• PCR Samples

Procedure

1. Using a balance, mass out 0.5 gram of agarose (for 1% gel, for 2% mass out 1.0 gram). Mix this with 50 mL of TAE 1X Buffer.
2. Microwave the mixture, mixing by swirling intermittently, until all the agarose is dissolved and the mixture is homogeneous and clear (about 2 minutes).
3. Pour the mixture into a gel plate and insert a correctly-sized comb (8, 10, etc. lanes). Allow to cool and solidify on the benchtop.
4. Mix your samples on a piece of parafilm. Use 10 μL of the appropriate DNA Ladder (1 kb, 100 bp, 2 log, etc.) combined with 1 μL SYBR Green/DMSO in the first lane. For all samples, mix 10 μl of sample and 2 μL of SYBR Green/Dye Mixture.
5. Place gel in tray into the gel electrophoresis apparatus - wells should be close to the negative (conventionally black) end. Fill apparatus with 1X TAE until the gel is completely submerged. Do not overfill - there is a guide sticker on the apparatus which marks the max fill line.
6. Load 10 μL of each sample into the appropriate wells.
7. Connect wiring and run gel electrophoresis at 110 V for 1 hour.
8. Analyze gel in UV light and see if samples match the expected size using the ladder as a guide.

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Phosphorylation

Materials

• PCR product that are to being ligated
• Ampligase thermostable DNA ligase reaction buffer
• T4 polynucleotide kinase[10 U/μL]
• 10 mM ATP
• Molecular Grade Water

Procedure

1. Make the following reaction mixture.

Component	20μl reaction
Molecular Grade Water	Calculated. Use to make reaction total volume = 20ul
PCR products	90 fmol each
Ampligase reaction buffer	2 μL
T4 polynucleotide kinase	1 μL
10 mM ATP	2 μL

2. Incubate reaction at 37 C for 1 hour.
3. Heat inactivate reaction at 65 C for 20 minutes.

Ligase cycling reaction

Materials

• Phosphorylated mixture
• Bridging oligonucleotide
• Ampligase thermostable DNA ligase reaction buffer
• Ampligase thermostable DNA ligase
• Molecular Grade Water

Procedure

1. Make a mixture of all bridging oligonucleotide, by adding 10 μL of each 100 μM bridging oligonucleotide stock and then adding Molecular Grade Water until 1000 μL.
2. Make the following reaction mixture.

Component	10μl reaction
Molecular Grade Water	Calculated. Use to make reaction total volume = 15ul
Phosphorylated mixture	11.60μl
Ampligase reaction buffer	1.5 μL
Bridge mixture	1 μL
Ampligase	.9 μL

3. Use thermocycler at folowing settings.

Cycle Step	Temp(C)	Time	Cycles
Initial denaturation	94	2 min	1
Denaturation	94	10s	25
Annealing	55	30s	25
Extension	66	60s/kb	25
Hold	4	Hold	1

4. PCR products are ready to be transformed.

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