Team:Brown/Notebook weekly Logs/Weekly Team3 Notebook

From 2009.igem.org

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S.epidermidis and Secretion Notebook
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=S.epidermidis and Secretion Notebook=
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07/16/09 - 07/23/09
07/16/09 - 07/23/09
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*Date: 07/16/09
*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.
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*Date: 07/17/09
*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.
**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.
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*Date: 07/19/09
*Date: 07/19/09
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*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:
*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:
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50ul Reaction
 
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50ul Reaction
*NEB2 Buffer/ Multicore = 5ul
*NEB2 Buffer/ Multicore = 5ul
*pSB1A3 DNA (700ng) = 15ul
*pSB1A3 DNA (700ng) = 15ul

Revision as of 19:15, 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.


  • Date: 07/19/09
    • We are ligating Signal Peptide insert into pSB1A3 according to the following:
    • Ligation with a total volume of 10ul:
      • Linearized DNA and semi vol vector = 2 – 4 ul
      • 1x Ligase Buffer
      • T4 DNA Ligase = 200ul
  • 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

  • NEB2 Buffer/ Multicore = 5ul
  • pSB1A3 DNA (700ng) = 15ul
  • 100x BSA = 0.5ul
  • EcoRI = 1ul
  • SpeI = 1ul
  • dH2O = 27.4ul
  • 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.
  • Date: 07/20/09
  • Notes from ADrian

Flowchart for Digestion, Ligation, and Transformation


<PHOTO HERE>


  • Date: 07/20/09- Mathing It Out, Take 1!
  • Ligation of Signal Peptide: BioBrick Into BioBrick Vector
  • 10:1 ratio
  • 4.4ul vector DNA of 11.4ng/ul
  • 0.4ul SigPep insert
  • 3 x [insert length (bp) / vector length (bp)] x Vector mass (ng)
  • 3 x [131bp / 2157bp] x 50ng = 9.1ng insert
  • 1ul 10x Ligase Buffer
  • 9.1ul H2O
  • 0.5ul Ty DNA Ligase
  • 5:1 ratio
    • 4.4ul vector DNA of 11.4ng/ul
    • 1ul SigPep insert
      • 3 x [insert length (bp) / vector length (bp)] x Vector mass (ng)
      • 3 x [131bp / 2157bp] x 50ng = 9.1ng insert
    • 1ul 10x Ligase Buffer
    • 8.5ul H2O
    • 0.5ul Ty DNA Ligase
  • 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


  • Date: 07/21/09
    • 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).
  • Date: 07/22/09- The Plan and the Execution!
    • 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
    • SpeI and XbaI are compatible restriction sites.
    • Here is our ideal plan:
      • SP
      • GFP


    • 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 .
    • PfuTurbo PCR of full Agr Operon:
      • First, resuspend Rev_Agr_EagI (19.43nm) in 194.3ul H2O to 100uM.
      • Dilute to 0.4uM
        • 100x =0.4 (10)
        • x = 0.4ul primer with 8.96ul H2O.
      • Per Reaction:
        • dH2O- 41ul
        • 10x cloned Pfu reaction buffer- 5ul
        • dNTPs at 10uM- 1ul
        • DNA template at 60ng/ul / 42.3ng/ul- 1ul
        • Primer 1- 1ul
        • Primer 2-1ul
        • PfuTurbo- 1ul
      • Transformed:
        • RFP (Kan)
        • GFP (Amp)
        • LacZ - (Kan)
      • 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.


  • Date: 07/23/09- Mathing It Out, Take 2!
    • 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.
    • Calculation for making SMMP [ Total volume = 100ml]

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

      • 1mol /L = X/ 0.055L
      • X12 = 18.8263g

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

      • 0.04mol/L = X/0.055L
      • X = 0.2554g

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

      • 0.04mol/L = X/0.055L
      • X3 =0.0022mol
    • 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:

    • Summary of SMMP

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

    • Redid glycerol stock for PSB1A3 – RA.
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