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 !!

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 (J4500x) Ligations

We'll do 5 ligations:

• 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)

General ligation considerations: 10μL total volume, about 50ng of each DNA

BSMT Ligation

• 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 BSMT (50ng)
• 2μL cut backbone (50ng)

Control Ligation (Barry's biobrick part)

• 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 BB part (50ng)
• 2μL cut backbone (50ng)

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