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

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

1. • 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

1. • 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