Team:Brown/Notebook weekly Logs/Weekly Team3 Notebook

(Difference between revisions)

Revision as of 19:13, 21 October 2009

S.epidermidis and Secretion Notebook

07/16/09 - 07/23/09

Date: 07/16/09
- We are going to use pSB1A3 instead of PSB1C3 (Ampicillin resistance instead chloramphenicol resistance). We are transforming cells with them.

Date: 07/17/09
- We miniprep the DNA from the overnight liquid cultures of pSB1A3-RFP. Now, we are digesting pSB1A3 with EcoRI and SpeI according to the same protocol from 07/15/09). We are using 11ul of SB1A3 plasmid DNA (500ng at 6.2ng/ul) to make 20ul of double digests. We are adding 3ul of dH2O to the digest. However, the gel extraction fails because too much gel (1.2g) was cut out.

The gel extraction’s concentration turns out to be too low so we are redoing the digest of pSB1A3 with EcoRI+ SpeI using Tom Knights Protocol:

50ul Reaction

We run the digest program on Thermal Cycler. We are incubating the digest at 37oC for 10 hr, then at 80 oC for 20min, and at 4 oC forever or until we need it.

Flowchart for Digestion, Ligation, and Transformation

We have desgined the primers for Trg/Envz:
Note: They must be digested (can be either PCRed by Tag or PfuTurbo).
Name: GC, Time, Length
- TrgF: 50, 65.3, 32
- TrgR: 37.5, 61.3, 32
- EnvzF: 48.1, 63.9, 27
- EnvzR: 59.3, 71.0, 27

- Colonies were picked from E.coli that were transformed with ligations of pSB1A3 + SP, and we started miniprep culture; insert: vector ratio of 3:1 worked best for transformation (the result for that ratio was about 5x as many colonies as 1:1 and 6:1).

- Here are results of the miniprepped pSB1A3-SP DNA:
- Insert: Vector Ratio DNA Concentration (ng/ul)
  - 3:1 69.0
  - 6:1 94.5

- We have 2 digestions running:
- 1) pSB1A3-SP with SpeI and PstI
- 2) SB1A2-GFP with XbaI and PstI

- We have 3 tubes that, due to a labeling mishap, might have the GFP/protein in it.
- We will digest and run all 3 out on a gel with the digest SB1A3-SP, and try to identify GFP by size.

- Double Digest using the protocol on 7/18/09 [Total volume = 50ul]
  - Mystery 1: 40ul DNA (at 16ng/ul) with 2.5ul H2O- 640ng DNA
  - Mystery 2: 30ul DNA(at ~30ng/ul) with 12.5ul H2O- 900ng DNA
  - Mystery 3: 15ul DNA (at 65.5ng/ul) with 27.5ul H2O 982.5ng DNA
  - pSB1A3-SP: 10ul DNA(at 94.5ng/ul) with 32.5ul H2O 1ng DNA

- The digests fail, but we are able to identified Mystery 1 and 2 as GFP (possibly because the band was about the same size as pSB1A3-SP). Now, we are growing overnight liquid culture of GFP cells. Also, we are growing plates of GFP, RFP, and LacX .

- - From the plate, we pick a colony containing SigPep – PSB1A3 ligation, and we prepare a glycerol stock for it and it is growing at 37oC overnight (12-16hr). Tomorrow, this glycerol stock will be freeze at -80 oC.

- The glycerol stock for SigPep – PSB1A3 ligation that we made yesterday has been put into the -80 oC freezer. We have plated the glycerol stock solution of pSB1A3 – RA on the LB+Amp plate to test whether or not the glycerol stock works. We found out that the glycerol stock volume is less than we expected so we decide to make another glycerol stock for PSB1A3 – RA.

1) 5.5 parts (55%) SMM Buffer = 55ml: a. 1M Sucrose (Molar mass = 342.29648g/mol)

b. 0.04M Maleic Acid (Molar mass = 116.1g/mol)

c. 0.04M MgCl2 (Molar mass = 95.211g/mol)

- To get 0.0022mol of MgCl2 from Magnesium Chloride Hexahydrate (Molar mass = 203.31g/mol)
- X4 = 0.4473g
- Put 18.8263g

2) 4 parts (40%) 7% Panassy Broth = 40ml: 3) 0.5 parts (5%) 10% BSA = 5ml:

1) 5.5parts SMM Buffer a. Sucrose- 18.8263g b. Maleic Acid- 0.2554g c. MgCl2 x 6H2O- 0.4473g d. ddH2O- Bring up to 55ml

2) 4 parts 7% Panassy Broth a. 100% panassy Broth- 2.8ml b. ddH2O- Bring up to 40ml

3) 0.5 parts 10%BSA a. 10x BSA- 50ul b. ddH2O- Bring up to 4.95ml

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 /16/09 - 07/23/09
 *Date: 07/16/09
- **We are going to use pSB1A3 instead of PSB1C3 (Ampicillin resistance instead chloramphenicol resistance). We are transforming cells with them.
+**We are going to use pSB1A3 instead of PSB1C3 (Ampicillin resistance instead chloramphenicol resistance). We are transforming cells with them.
 *Date: 07/17/09