Experiment No 3

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

Personnel:

Choon Kit, Hung, Dr Tan TL

Title of Experiment:

Experiment could be classified in 3 phases:

I.	Preparation for Punch Tool Cleaning
II.	Paper Punching of  Biobrick
III.	Transformation

Materials:

Phase I:
8 x 2.0ml conical bottom tubes
3 x weighting tray
Kimwipes 
Blotting paper (back of binder)
10% bleach (chlorine)
DI water
95% ethanol
Pippette with pipette tips

Phase II:
TE (10:1, pH 8.0)
Olfa Cutting Mat (back of binder)
Punch Tool
0.5ml PCR Tubes
Pippette and Pipette Tips

Phase III:
Spots soaked in TE
2.0 ml conical bottom tubes (one per spot)
Ice
Competent cells
42oC water bath / 37oC incubator
LB or SOC broth
Agar Plates with appropriate antibiotic (Ampicillin in this case)
Transformation control DNA (e.g.  PUC18 plasmid)

Protocols/Procedures:

Phase I:

1. To 8 conical bottom tubes, 3 of them filled with 1ml of 10% bleach each, 3 filled with 1ml of DI water, and the last 2 filled with 1 ml of 95% ethanol.
2. Practice punching using blotting paper before beginning experiment.
3. When after each punch of plasmid, the puncher is washed by tipping into the contents of the 8 conical tubes in a weighing tray in the following sequence: 3 times bleach, 3 times DI water, 2 times ethanol.
4. Blot with a Kimwipe (high grade tissue) and allow to dry for 5 minutes before punching another spot of plasmid.

Phase II:

1. Pipette 5ul of TE buffer to 50oC in PCR Eppendorf tubes. Warm a water bath to 42oC
2. Using the cutting mat as a backing, a sample of plasmid is punched off the edge of the spot using the puncher by pressing firmly down and rotating the punch tool. (Do not press the ejection button)
3. Eject the punched paper spot into the PCR tube by pressing the ejection button
4. Clean punch tool to prevent cross contamination as mentioned in phase I

Phase III:

1. Soak the spots in 5ul of warmed TE for 20 minutes. Also, thaw the competent cells on wet crushed ice.
2. Chill labeled 2ml concial bottom tubes on wet ice. Add 2ul of DNA in TE and 50ulof thawed competent cells to the tubes. Extra eluted DNA can be kept frozen at -80oC for several weeks.
3. Also, do a control transformation using pUC18 plasmid DNA.
4. Hold the DNA and competent cells on ice for 30 minutes to increase transformation efficiency.
5. Heat shock the cells by immersion in a pre-heated water bath at 42oC for 1 minute. This is to improve heat transfer to the cells.
6. Incubate the whole floating tray of conical tubes on ice for 2 minutes.
7. Add 200ul of LB broth. (no antibiotic, check that broth is not turbid, which indicates contamination and bacterial growth)
8. Incubate the cells at 37oC for 1-2 hours. 2 hours is optimal for plasmids with other antibiotic resistance other than amipicillin.
9. Label an LB agar plate with amipicillin with the date, project name, and plasmid e.g. LBA – 26May08 – iGEM – Fe promoter, LBA – 26May08 – iGEM – LacI, LBA – 26May08 – iGEM – pUC18.
10. Plate 250ul of the incubated cell culture on the plate
11. Incubate the plate at 37oC overnight or 12-14 hours. Do not incubate too long as the antibiotic may break down, resulting in growth of untransformed cells.

Observations:

2 colonies observed in LacI LB plate, 0 colony in Fe2+ LB plate, positive control pUC-18 showed many colonies.

Conclusion:

Fe2+ not successfully transformed in home-made competent cells. LacI and pUC-18 transformation were successful.

Notes:

Refer to video clip 26 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.