Team:IvyTech-South Bend/Notebook
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Today we will electroporate Agrobacterium with part T9002 using protocol from pg 22 – 23 | Today we will electroporate Agrobacterium with part T9002 using protocol from pg 22 – 23 | ||
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== 9/22/10 == | == 9/22/10 == |
Revision as of 13:48, 12 October 2010
Discussion
Contents |
Notebook
June | ||||||
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7 | 8 | 9 | 10 | 11 | 12 | 13 |
14 | 15 | 16 | 17 | 18 | 19 | 20 |
21 | 22 | 23 | 24 | 25 | 26 | 27 |
28 | 29 | 30 |
July | ||||||
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5 | 6 | 7 | 8 | 9 | 10 | 11 |
12 | 13 | 14 | 15 | 16 | 17 | 18 |
19 | 20 | 21 | 22 | 23 | 24 | 25 |
26 | 27 | 28 | 29 | 30 | 31 |
August | ||||||
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2 | 3 | 4 | 5 | 6 | 7 | 8 |
9 | 10 | 11 | 12 | 13 | 14 | 15 |
16 | 17 | 18 | 19 | 20 | 21 | 22 |
23 | 24 | 25 | 26 | 27 | 28 | 29 |
30 | 31 |
September | ||||||
M | T | W | T | F | S | S |
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6 | 7 | 8 | 9 | 10 | 11 | 12 |
13 | 14 | 15 | 16 | 17 | 18 | 19 |
20 | 21 | 22 | 23 | 24 | 25 | 26 |
27 | 28 | 29 | 30 |
October | ||||||
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4 | 5 | 6 | 7 | 8 | 9 | 10 |
11 | 12 | 13 | 14 | 15 | 16 | 17 |
18 | 19 | 20 | 21 | 22 | 23 | 24 |
25 | 26 | 27 | 28 | 29 | 30 | 31 |
November | ||||||
M | T | W | T | F | S | S |
1 | 2 | 3 | 4 | 5 | 6 | 7 |
8 | 9 | 10 | 11 | 12 | 13 | 14 |
15 | 16 | 17 | 18 | 19 | 20 | 21 |
22 | 23 | 24 | 25 | 26 | 27 | 28 |
29 | 30 |
8/24/10
Today we will be pouring new plates for streaking new transformed cells.
8/27/10
We will be running a gel to determine if our DNA sample was successfully electroplated.
8/27/10
Today we will be electroplating part KBBa_3131010
8/31/10
Results of transformation - the plates had growth but (no) distinct colony pattern.
8/31/10
After agar is dissolved, and allowed to cool, I will add Amp in different doses to specific plates. JH
9/1/10
Today the team will be making up the same plates as I made yesterday. Professor T. wants new Amp stock made to document how much is used into the stock.
9/2/10
Results from streaking –
9/2/10
Protocol For Making LB-Broth/Agar
9/3/10
Today I’m finishing making the LB/Agar from yesterday.
9/9/10
Due to conflict we will be changing from E Coli to yeast.
9/10/10
Lux Casette Right Promoter BBa_I1051
9/14/10
Today we found growth from our Electroporated E – Coli parts
9/15/10
- Today we will be extracting DNA from out electroporated cells/T9002
9/16/10
Today we will wash our electrocomp grown t/pos bacteria with 1.2 mL Glycerol then electroporate Following protocol from page 15 First prof. T sterilized the glycerol passing it through the .2 micron filter into 50 mL sterile centrifuge tube 1 – then we added 1 mL 10 percent Glycerol to suspended cells 2 – then centrifuged at 1000 rpm for 10 min - then removed supernate with BR 1000 micropipettor - then add 1 mL 10 percent Glycerol then repeat steps 1 & 2 “4” times then remove sup. and freeze cells at -80 degrees C freezer.
- Today we also set up a new IGem work are in back lab look at pic
9/17/10
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.
9/21/10
Today we will electroporate Agrobacterium with part T9002 using protocol from pg 22 – 23
9/22/10
Today we have growth from electroporation done yesterday we are going to test by hitting with (AHL) the 20 mL grew not 5 mL sample
9/22/10
The only one that should glow should be (C)/(3) E – Coli w/transformed part in LB For Supernate of A.Tumafaciens
9/23/10
Today I have been setting up for the trial of our part T9002 inside agrobacteria to see if in the presence of AHL it floureces.
9/24/10
Today I’m testing absorbance of our trials @ 395nm
9/28/10
Today we will be picking 2 colonies from the Lacz, LuxR electroporated plates that were streaked on 9/22/10
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