Transformation Test for Ampicillin

1) Thaw on ice. Add 5 micro of Dna (one ligation one control) sit for 30 min.

2) Heat shock at 42C for 30 seconds.

3) Add 950 uL of SOC (from fridge).

4) Spread 200uL of cells and ligation mixtures onto the plates.

5) Incubate overnight at 37C.

cPCR, chloramphenicol, and culturing protocol

1) Pipette 10 µL of molecular grade water into 2mL tubes.

2) Circle a colony and label it with a number to use.

3) Use the pointy end of the inoculating loop to pick up a colony of bacteria and swish around in the water.

4) Pipette 15 µL of molecular grade water into PCR tube with pre-made master mix bead. Remember to pipette on the side of the tube without touching the bead. Allow bead to dissolve into the water.

5) Take 5 µL of your water-bacteria mixture and transfer it to the PCR tube with the 15µL of water.

6)Pipette 2.5µL of each primer (total of 5µL for both forward and reverse primer) into the PCR tube.

7)Label the PCR tube with the same number you picked for your bacterial colony and place into thermocycler.

Prepping Plasmids

1)Pellet 1-5mL bacterial culture (centrifuge for 30 seconds). Discard supernatant.

2)Resuspend pellet in 200uL Plasmid Resuspension Buffer(B1). (Vortex or pipet to ensure cells are suspended.

3)Prep the next step before conducting this step!!!!Add 200uL Plasmid Lysis Buffer (B2) gently invert tube 5-6 times.and incubate for 1 minute at room temp. DO NOT VORTEX!!!!!!

4)Add 400 uL of Plasmid Neutralization Buffer (B3), gently invert until neutralized and incubate at room temp for 2mins. DO NOT VORTEX!!!!!

5)Centrifuge lysate for 2-5 mins.

6)Transfer supernatant to the spin column and centrifuge for 1 min.

7)Re-insert column and add 200uL of plasmid wash buffer 1. Centrifuge for 1 min.

8)Add 400uL of plasmid wash buffer 2 and centriuge for 2mins.

9)Transfer column to a clean 1.5mL microcentrifuge tube. Discard the flow throug, and do not allow the tip of the column to touch the flow through.

10)Add 50uL of DNA Elution Buffer to the center of the matrix. Wait for 1 minute then spin for 1 minute to elute DNA.