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From 2010.igem.org

Established Team lab manual

Contents 1 8/27 BBa_K131010 part - plasmid purification 2 cont... 3 For Transformation 4 For Electrophoresis

8/27 BBa_K131010 part - plasmid purification

We are in pursuit of the K131010 part. An initial transformation was performed buy a single colony was not preserved for a glycerol stock.

A broth culture of the original fusion was preserved and plasmid DNA was isolated after 48 hr. culture.

Followed Quantum Prep Plasmic Miniprep Kit.

1. 2x Pellet of over weekend culture from transformation*

2. resuspend 200x cell resusp buffer

3. 250 microl cell lysis

4. 250 microl neutralization solution

5. Pelletid 5 min x 2 (because pre was not pelleting)

6. Transferred sup to spin filter

7. Added 200 microl matrix suspend

8. Wash x2 w/ 500 microl

9. Elute 100 microl H2O (ultrapure)

10. Xfr. to icewater

11. Froze to -20 C small freezer.

cont...

As noted the plasmid containing bacteria was contaminated with the possibly Amp resistant bacteria

Today

1. retransformation of E. coli w/ the purified DNA from "K1311010 purif 8/17/10 GT by JH

2. Agarose gel electrophoresis of purif. DNA above

For Transformation

Competent cells thawed from -80 c on ice

Boy "IGEM electro competent cells 7/23/20 GT

SOB Media 6/29/10

Electroporation according to Richard lab (open wet ware) except 1 ml prewarmed SOB added directly to electorporation cuvette, cells removed to controls A & B.

Control A - 1 vial competent cells remove 10 microl and 190 micrl SOB > spread on 100 microl plates on

Control B - Mock elctroproate remainder of the cells, remove from cuvette + add to 100 microl SOB.

K121010 ELECTR0- Electroporation ao micrl purif DNA to 1 new vial elec. comp. cells

Electroporation data

2nd pulse 1.80 KV 4.2 ms Control B

2nd pulse 1.80 KV 1.7 ms transformation w/ K131010

For Electrophoresis