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From 2010.igem.org

Today we will be doing a DNA Extraction from/of part T9002 for electroporation this will determine if we have a stable host for our beast this will also Work!

Following protocols from pgs 15 &19 -instead of Electroporating our part we will use PG10 to determine if these lines are good hosts

First we will be pouring a full pack of LB/Agar/Amp plates using the LB/Agar I made on 9/2/10 then adding amp made by DG on 9/1/10 we will also add 5 mL Arabonse to the Agar before pouring plates have been poured now letting stand for an hour to harden.

-Now I will be making 1L of LB/Broth for Culturing. Lot 9082989 ex. 2014-02-28 BD Difco LB/Broth – Lennox 20 g per 1L so I will put 10 g into 2 elenmyier flask/capped one weighed 10.0023 g and the other weighed 10.0011 g then I added 500 mL into each flask then shook to mix then dissolved in microwaved for 8 oz setting once then placed into Autoclave for 15 min @ 121 degrees C until done.

Now we are going electroporate our grown t/pos Electro Comp Cells from yesterday with PG10 using protocol from pg 15 changing # 5 to 5 mL of PG10 We will be using a different protocol for electroporation of grown t/pos bacteria from Bio-Rad’s website prof. T will print and I’ll place here.

The mycobacterium will be done by electroporation protocol on pg 15.

1) First I placed a Cuvette into Ice bath and thawed electro comp cells

2) I set up 5 1.5 mL Sterile Centrifuge tubes with 900 mL SOB media

3) we will electroporate Irene’s electrocomp cells w/PG10 and mine will contain the parts after electroporation.

Code AT – A.tumefacions SL – S.lactis BM – B.magetanium MP – Mycobacteria BT – B.thurigensis

split cells using 40 mL Hy part my cells then electroporate

I electroporated all except mycobacteria now letting cells sit for at least Just added 1 mL to electroporated Cultures they had sat for 20 min before adding.

- Now I will electroporate mycobacteria using E – Coli protocol from pg 15 using 40 mL cells 4 mL pg10 Electroporation hit twice Read out 1.8 kv @ 5.2 ms then 1.79 kv @ 5.1 ms. then placed into 900 mL SOB media then incubate @ 30 degrees C for 3 hrs.

Results From this we found that agrobacteria will be the best host for our beast/IGem we will do further testing towards our ultimate goal.