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From 2010.igem.org

Contents 1 9/22/10 1.1 Electroporation of DNA (LuxR) and (LacZ) 1.2 Electrophoresis of new ligased DNA 1.3 Ligation protocol

9/22/10

Hit it all with AHL Scarape baterian the resuspend in 1 ml lb. (bacteria was grown by jh) Cuvettes A_ 200 ul of bacteria + 800 ul of lb B_ 200 ul of bacteria + 800 ul of A. tumaflens supernant C_ 200 ul of bacteria + 800 ul of e.coli supernant D_ 200 ul of bacteria + 800 ul of lb E_ 200 ul of bacteria + 800 ul of A. tumaflens supernant F_ 200 ul of bacteria + 800 ul of e.coli supernant

Things we need to do -streak isolate on LB/AMP from transorm plate.

- Make agrobacterium competent cells by washing 3 time in 10% glycerol - Transform I 732094 (LacZ) - - Purify DNA 091107 (pLux) - Transform e.coli F2620 (LuxR) - Jh too sample of Agrobacterium + Electrocom cells + T9002 DNA. - Then he streaked colony on plate and placed in incubator - I pulled part I732094 (LacZ) from well 5ul then added to steril tube containing 50 ul electorcomp cells - I then pulled part F2620 (LuxR) from well by adding 10 ul of di H2O then pulling 5ul of di H2O and DNA(LuxR)

Electroporation of DNA (LuxR) and (LacZ)

1.79 kv @ 5.4 ms

1.80 kv@ 5.3 ms

-pulsed twice (LuxR is first

- susan added 900 ul of SOB media to cuvette

Then pulled 900 ul of SOB+electroporated DNA+cells

Then added to sterile tube 3:30 pm

Electrophoresis of new ligased DNA

microwave agrose gel to liquefy

we added 4ul of ithidium bromide to a 50 cc tube

Note: we used extra precaution of ithidium bromide and used biohazard bag for disposal

we added 40 ml of agrose gel into taped gel box

I made another 1x buffer 50l

490 ml of 18ohm H2O and 10 ml of TAE Buffer

put gel into Electrophoresis box and poured Buffer into Box

I’m going to set up my DNA and loading dye

Ligation protocol

1. Add 1 lul ofdH20

2. Add 2ul from each sample you will be ligating (destination plasmid, and part)

3. Add 2ul ofT4 DNA Ligase Reaction Buffer

4. Add 1 ul of T4 DNA Ligase

5. Mix well, and spin down.

6. Incubate for 30min at 16C and 20min at 80C to heat kill.

7. Use 2ul of ligation te transform into competent cells.

combination of digests (w/ IF)

- A B C D

L10 X X

L30 X X

T10 X X

T30 X X

Note: We have to recut T9002 (30 ul) w/ the P enzyme only ( The new T9002)

Irene is using the T9002 from 10/18/10 (Blue Macker)

I added 1ul of PS-I to my 30 ul of restricted /cut T9002 (cut) + P into thermocycle 2 our Tet backbone cut w/ P and E.

Thermocycler set for 30 min @ 37C and 20 min @ 80 C, with larges vol of 50 ul.