"

From 2010.igem.org

Contents 1 10/1/2010 1.1 BBa_pSB1T3—Tetracycline resistant plasmid—Plate 1 Well 7A 1.2 electrophoresis 1.3 Electroporation Comp E-Coli w/PGlo 2 9/24/10 3 10/1/10

10/1/2010

We are going to gram stain the agrobacteria, E-Coli, and florescent spl. and efflorescent spl. of agro to determine if we have agro that we are working with.

Hun Young is stetting up this gram stain placing 4 circles on a slide 0 X 1 2 and following the Gram Stain Protocol.

0-has shown under oil immersion was groups of red rods

X-has shown lest clusters but under oil shown was red rods as well

1-

2-

BBa_pSB1T3—Tetracycline resistant plasmid—Plate 1 Well 7A

Today we are extracting a plasmid following protocol for extraction.

1) added 250 ul resuspension sol. and sightly vortexed to mix well.

2) added 250 ul of lysis sol. to 1.5 ml centrafuge tube with resuspended cells.

Following the protocol exact to the letter. I;m doing E-Coli w/ T9002

After DNA purification we drew out 5 ul from each spl. and placed into new tubes for electrophoresis, which we will do after lunch.

Placed remainder (45ul) of each in plastic frezer box in -80C freezer

electrophoresis

I will be loading our electrophoresis gel (all with loading tip from sterile box)

Well 1- Ruler

Well 2- T9002 with 5 ul loading dye

Well 3- F2620 with 5 ul loading dye

Well 4- I732094 with 5 ul loading dye

Well 5- Ruler II

Note: Gel contains lots of bubbles may be source of error. The gell turned out to show that the LacZ and LuxR had about the same size plasmid and F2620 had a much smaller plasmid which is basically what we were expecting after taking pictures, we bagged and froze.

Electroporation Comp E-Coli w/PGlo

Now I will be electroporating Comp E-Coli cells with PGlo then plate right away to allow to grow them.

taking and growing new Ecoli Comp Cells made by Biolabs inc. I will be taking 40 ul from main tube and placing into 4 new tubes then shoodting LB-Lennox into the main tube and placing into 15 ml centrifuge tube with LB and Amp to grow all weekend.

1) thawing cells in an ice bath along with .2cm cuvette then electorporating E-Coli w/PGlo. Following Electroporation protocol

Incubated 10 min. then plated on Amp/Arab LB-Lennox

9/24/10

DG

1-pull sample of 500 ul of A. Tumefaciens , T9002 and A. Tumefaciens only w/ sterile loop and suspend in 1 ml of broth separately (microfuge tubes steril) (LB Broth made by JH 9/17/10)

2- Pull 10ul of agro+T9002 and 500up agrao sub and 500 ul of LB Broth and add it to a steril tube. - I’m going to do this procedure 2 more times (A-C) 3- Next I’m ging to pull 10 ul of A.Tmefaciens (only) Sample. (the one I pulled from platae and suspended) then I’m going to pull 500 ul of LB Broth and 500 ul of e.coli sup. And add all three to sterile tube. - Im going to do the same proc 2 more times (D-F) 4- I’m then going to get 3 sterile microfuge tubes and ad 1 mil of LB broth (same broth from prev steps) to cach tube. Then I’m going to add 10 ul of E.coli sup (same from prev steps and 10 ul of Agro-T9002 (same sample that I Pulled from plate) 5- Then put all the tubes A-I inot the small 30C dry incubator @ 3:30 pm 9/24

10/1/10

Transfer/Suspension Assay Trial 3

I filtered the E.coli sup using a sterile filter syringe.( Put into sterile 45ml tube)

• I then added 1ml of LB Broth (made by JH) to 45 ml sterile tube containing concentrated A. Tumefaciens with part T9002.

• Then pulled 10 ul of A.Tume. plus T9002 LB/Broth and put it into sterile cubette.

• Spec. read blank at 0.064

• Spec. read my sample at 0.052

• I then took 4ml of A. tume. sup. made (by brandi) and filtered into sterile tube.

• Spec. read 0.023 for the A.tume that I pulled from a plate and suspended in 1ml of LB Broth

• I pulled 1.07 ml of LB/Broth and added that to the 1ml of A.Tume. suspended in LB/Broth

• I then pulled 10 ul of my A.Tumefaciens colony and resuspended in my 2.07 ml of LB/Broth + A.Tume.

- (1ml)(0.05OD)/ 0.043/OD  ; (1ml)(0.05OD)/ 0.04/OD (1.2 ml)

• I need to add more cells b/c my OD reading is less

• I’m going to pull more cells from (A.Tume) the plate to add to my LB/Broth+A.Tume.

• Spec reading after adding more cells, 0.030 OD.

• not sure why the OD reading is decreasing(?)

• calibrated machine put the blank in the “B” section of spec.

• respect of my sample (0.023 OD)

--for dgarveyRchamberlin 13:04, 27 October 2010 (UTC)