Experiment No 3

Date 27-28 May 2008 
Start Time 1530						
End Time 1800

Personnel:

Choon Kit, Hung, Dr Tan TL

Title of Experiment:

The experiment could be classified in 3 phases

1.	Amplification of transformed colonies (for 2 samples)
2.	LacI Plasmid Extraction using Qiagen MiniPrep Kit
3.	Plasmid analysis by Gel Electrophoresis

Materials:

Phase I:
2 x 15ml centrifuge tubes
Marker
LB broth with amipicllin resistance
2 x inoculating loops
Agar plate containing transformed colonies

Phase II:
Qiagen MiniPrep Kit
Mega Centrifuge
Small Centrifuge
Pipette with pipette tips
2 x 1.5ml centrifuge tubes
2 x 2.0ml centrifuge tubes 

Phase III:
PstI and EcoRI in icebox
BSA and EcoRI buffer 
DI water
2 x PCR tubes
Pipette with pipette tips
DNA marker (10kb)
Blue loading dye
Gel Electrophoresis setup

Protocols/Procedures:

Phase I:(done in 27 May)

1. Label the 2 tubes with LacI(1) and LacI(2) on cover and sides.
2. Sterilized LB Bottle before bringing into the fume hood with 70% ethanol solution. Transfer approximately 20 ml of LB broth into one cryotube and cover the LB Bottle. Transfer another 10 ml of LB broth from one cryotube to the other.
3. Select 1 colony using inoculating loop by gently touching the tip with the Agar so that the colony just sticks to the tip of the loop.
4. Carefully insert the tip of the loop into the tube with LB and mix uniformly by rubbing the tip of the loop with the tube’s bottom.
5. Incubate at 37oC overnight with vigorous shaking.

Phase II:

1. After incubating overnight, centrifuge the 2 tubes from phase I using a Mega centrifuge. The machine is settled at temperature 4oC, Rotor 11133, maximum 5500 RPM, time 10min. Check settings and set to ‘lock’ mode.
2. Empty the supernatant from both tubes and invert to dry remnants of liquid from the tubes. This is to ensure the bacteria pellet is as dry as possible.
3. Resuspend pelleted bacterial cells 250ul of buffer P1 and transfer to a micro centrifuge tube. The bacteria are resuspended by pipetting up and down several times until no cell clumps remain. Keep buffer P1 at 4oC after use.
4. Add 250ul of buffer P2 and mix gently by inverting the tubes 6 times. Do not allow lysis to exceed 5min.
5. Add 350ul of buffer N3 and mix thoroughly by inverting the tubes 6 times. Solution should turn cloudy.
6. Centrifuge the tubes for 10 minutes at RPM of 13200.
7. Gently pour the supernatant from step 4 to the QIAprep spin column. The plasmid DNA is now trapped within the membrane of the QIAprep column. Empty the filtrate and dry by inverting and tapping on tissue. Centrifuge for 1 minute at 13200 rpm.
8. Add 750ul of PE buffer to wash the QIAprep column. Leave to stand for 5min. Spin for 1min at 13200rpm, then empty the filtrate and spin again. The outer tube of the QIAprep can be thrown away.
9. Place the inner QIAprep column into clean 1.5ml micro centrifuge tube. Elute the plasmid DNA by adding 30ul of buffer EB just near to the inner membrane. Allow to stand for 1 minute, then centrifuge for another minute. 30ul is just sufficient to cover the membrane cross sectional area.
10. Store LacI(1) and LacI(2) plasmid DNA at 4oC.

Phase III:

1. 2ul of EcoRI buffer, 0.2ul of BSA, 1ul of EcoRI, 1ul of PstI and 11.8ul of DI water into one PCR tube and do a pulse spin at 2000 rpm. Then transfer 8ul from the cocktail mixture into the second PCR tube.
2. Add 2ul of LacI(1) plasmid DNA to a PCR tube. Repeat for LacI(2). Centrifuge for a short pulse with rpm just reaching 3000rpm.
3. Incubate at 37oC for 2 hours (for preliminary analysis. For total ligation, allow incubation of at least 4 hours)
4. Prepare the agarose gel setup. Add 2ul of blue loading dye to 10ul of ligated sample in the PCR tube. Load 12ul of DNA marker, and ligated plasmid into each electrophoresis well.
5. Run the gel electrophoresis for 20min, ensuring the loading dye do not exceed the whole span of agarose gel.
6. Scan the agarose gel under UV.

Observations:

LacI operon 200bp corresponding to experimental gel electrophoresis

Conclusion:

Preliminary test indicated that the Miniprep was successful.

Notes:

Refer to video clip 27 May 08 and 28 May 08 for details.

To be follow up:

1. Repeat whole transformation of Fe2+ plasmid using commercially competent cells.
2. Pick out 2 colonies from LacI LB plate into LB broth with ampicillin overnight.