IGEM:MIT/2006/Notebook/2006-7-5

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Contents

BAMT PCR Product Eco/Pst Digest

BEGAN AT 11:15 AM

  1. We are cutting the BAMT pcr product (using EcoRI and PstI)--
  2. Total reaction volume is 50μL
  3. the amount of dna desired is about 800 nanograms
    • BAMT pcr product at 58.8 ng/μL -----add 13.6 μL
  4. we are also going to use Dpn1 only in BAMT cut reaction to destroy dna that is not a pcr product (i.e. get rid of extra template)
  1. BAMT pcr cleanup product cut reaction:
    • 28.9 μL water
    • 5 μL NEB buffer 2
    • .5 μL BSA
    • 13.6 μL BAMT pcr cleanup product (concentration = 50 ng/μL)
    • .75 μL Pst1
    • .75 μL EcoR1
    • .5 μL Dpn 1
  2. Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever.
  3. Did a PCR cleanup & nanodrop: concentration of digested BAMT = ??ng/μL
  4. run a gel before moving on to ligation !!

New Liquid Cultures for GC

Used 40 uL SA, 20 uL cell cultures, 10 mL LB Kan in each of the following tubes:

SAMT, not induced

BSMT, not induced

SAMT, induced

BSMT, induced

BSMT BB, induced

  • Note that the SAMT and BSMT cultures were two weeks old.

Measurement of Stationary Phase Promoter

Francois' rpos promoter was tested on a 96-well plate for fluorescence. The following wells were filled accordingly:

A1- 200 uL rpos promoter attached to GFP liquid culture

A2- 200 uL rpos promoter attached to GFP liquid culture

A3- 200 uL rpos promoter attached to GFP liquid culture

A4- 200 uL constitutive promoter attached to GFP liquid culture

A5- 200 uL constitutive promoter attached to GFP liquid culture

A6- 200 uL constitutive promoter attached to GFP liquid culture

A7- 200 uL LB media

A8- 200 uL LB media

A9- 200 uL LB media

We will read out the fluorescence data tomorrow.

ATF1 PCR Product Xba/Pst Digest

BEGAN AT 11:15 AM

  1. We are cutting the ATF1 pcr product (using XbaI and PstI)--
  2. Total reaction volume is 50μL
  3. the amount of dna desired is about 800 nanograms
    • ATF1 pcr product at 30.8 ng/μL -----add 26 μL
  4. we are also going to use Dpn1 only in ATF1 cut reaction to destroy dna that is not a pcr product (i.e. get rid of extra template)
  1. ATF1 Digest:
    • 16.5 μL water
    • 5 μL NEB buffer 3
    • .5 μL BSA
    • 26 μL ATF1 pcr cleanup product (concentration = 50 ng/μL)
    • .75 μL Pst1
    • .75 μL XbaI
    • .5 μL Dpn 1
  2. Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever.
  3. Did a PCR cleanup & nanodrop: concentration of digested SAMT = ??ng/μL
  4. run a gel before moving on to ligation !!

BACKBONE pSB1A3-1 bkb Xba/Pst Digest

BEGAN AT 11:15 AM

    • 35 μL water
    • 5 μL NEB buffer 3
    • .5 μL BSA
    • 8 μL backbone plasmid
    • .75 μL PstI
    • .75 μL XbaI

CONTROL (BSMT-BB) Xba/Pst Digest

    • 31.58 μL water
    • 5 μL NEB buffer 3
    • .5 μL BSA
    • 11.42 μL BSMT-BB (from BSMT miniprep @ 71ng/μL )
    • .75 μL PstI
    • .75 μL XbaI

Glycerols and Miniprep of BSMT biobrick

  1. Did minipreps of A, C, E BSMT liquid cultures to get the BSMT biobrick plasmid.
  2. Made glycerols of these cells (which contain the BSMT biobrick)

SAMT-BB (J45001), BAMT-BB (J45002), & ATF1-BB (J45006) Ligations

RUN GELS OF DIGESTS TO DOUBLECHECK THAT THEY WORKED!

Nanodrop readings following PCR clean-ups: (?? μL for 50 nanograms)

  1. EP cut pSB1A3-1 = 29.8 ng/μL (2 μL)
  2. EP cut SAMT = 10.4 (5 μL)
  3. EP cut BAMT = 13 ng/μL (4 μL)
  4. EP cut E0040 = 11.7 ng/μL (5 μL)
  5. XP cut pSB1A3-1 = 22 ng/μL (2.5 μL)
  6. XP cut ATF1 = 28.1 ng/μL (2 μL)
  7. XP cut BSMT = 15.7 ng/μL (3.5 μL)

We'll do 5 ligations, 10 μL total volume each:

  • EP cut pSB1A3-1 and EP cut SAMT
  • EP cut pSB1A3-1 and EP cut BAMT
  • EP cut pSB1A3-1 and EP cut E0040 (+ Ctrl)
  • XP cut pSB1A3-1 and XP cut ATF1
  • XP cut pSB1A3-1 and XP cut BSMT (+ Ctrl)


EP cut pSB1A3-1 and EP cut SAMT

  • 1.5μL water
  • 1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells strongly like wet dog!!)
  • .5μL T4 DNA ligase enzyme
  • 5μL cut SAMT (50ng)
  • 2μL cut backbone (50ng)

EP cut pSB1A3-1 and EP cut BAMT

  • 2.5μL water
  • 1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells strongly like wet dog!!)
  • .5μL T4 DNA ligase enzyme
  • 4μL cut BAMT (50ng)
  • 2μL cut backbone (50ng)

EP cut pSB1A3-1 and EP cut E0040 (+ Ctrl)

  • 1.5μL water
  • 1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells strongly like wet dog!!)
  • .5μL T4 DNA ligase enzyme
  • 5μL cut Barry's E0040 part (50ng)
  • 2μL cut backbone (50ng)

XP cut pSB1A3-1 and XP cut ATF1

  • 4μL water
  • 1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells strongly like wet dog!!)
  • .5μL T4 DNA ligase enzyme
  • 2μL cut ATF1 (50ng)
  • 2.5μL cut backbone (50ng)

XP cut pSB1A3-1 and XP cut BSMT (+ Ctrl)

  • 2.5μL water
  • 1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells strongly like wet dog!!)
  • .5μL T4 DNA ligase enzyme
  • 3.5μL cut BSMT (50ng)
  • 2.5μL cut backbone (50ng)


Ligate at room temp for 15 to 20 minutes and then proceed to transformation

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