Team:CSU Fort Collins/Experiments

Component	50μl reaction
Molecular grade H2O	Added first. 32.5μL 33.5ul for colony PCR
5x Phusion HF Buffer	10μL
10 mM dNTPs	1.0μL
Primer A (10μM)	2.5μL
Primer B (10μM)	2.5μL
Template DNA	1.0μL 0uLcolony PCR
Phusion DNA polymerase	Added last. 0.5μL

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

Measure concentration of clean-up PCR products with a nano drop
Run 10µL of clean-up PCR with 2ul of SybrGreen DMSO products on a gel 1%Agarose Gel (110V for 60mins). Look for How Many Base Pairs your insert is
Digest (Insert) and (Backbone )with ________+__________ at 37oC for 90mins.

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

Clean up all DNA fragments with PCR clean-up kit.
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
Incubate at 16C overnight on a heating block.

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

Transformation (Need 2x LB+Antibiotic plates. Check for plates. Check for hockey spreaders or glass beads):
- Set water bath to 42C
- Remove LB+Antibiotic plates from 4C and allow them to come to RT.
- Thaw chemically competent cells on ice. Leave in microcentrifuge tube.
- Add 5μL of ligation mix chemically competent cells.
- Add 5uL of digested back bone to chemical competent cells as a control
- Incubate on ice for 30 min.
- Heat shock cells for 60 sec at 42C without shaking.
- 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.
- Place tube horizontally in shaker. Incubate at 37oC and 225 rpm for 1 hr.
- In the laminar hood, spread 100uL of transformants onto LB+Antibiotic plates.
- Leave plates in 37C incubator overnight. Store remaining liquid cultures in 4oC.
Next day, pick and annotate (give each colony a #) 16 colonies onto new LB+Antibiotic plates. Place new plates in 37oC overnight.
Verify all 16 colonies via PCR with Taq DNA polymerase(DO NOT USE PHUSION!!!) using these primers:

Primer A:_________ Tm=__C

Primer B:_________ Tm=__C

Use the following thermalcycler protocol:

Cycle Step	Temp(C)	Time	Cycles
Initial denaturation	95	5 min	1
Denaturation	95	30s	35
Annealing	LowerTm+1	30s	35
Extension	68	41min/kb	35
Final Extension	68	5min	1
Hold	4	Hold	1

Run 10µL of all 16 PCR with 2ul of SybrGreen DMSO products onto 1% Agarose gel (110V for 60mins). Expected size-What is your inserts size????
PICK BEST TWO MUTANTS and start overnight cultures in 2mL LB + Antibiotic. Leave them to grow in the 37C incubator overnight.
Next morning; miniprep them (there should be 4 plasmids). Measure their concentrations with the nanodrop.
sequencing

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.
Microwave the mixture, mixing by swirling intermittently, until all the agarose is dissolved and the mixture is homogeneous and clear (about 2 minutes).
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.
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.
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.
Load 10 μL of each sample into the appropriate wells.
Connect wiring and run gel electrophoresis at 110 V for 1 hour.
Analyze gel in UV light and see if samples match the expected size using the ladder as a guide.

Experiments