IGEM:MIT/2006/Notebook/2006-7-5: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 64: Line 64:


'''Nanodrop readings following PCR clean-ups: (?? μL for 50 nanograms)
'''Nanodrop readings following PCR clean-ups: (?? μL for 50 nanograms)
#EP cut pSB1A3-1 =  
#EP cut pSB1A3-1 = 29.8 ng/μL (2 μL)
#EP cut SAMT =
#EP cut SAMT = 10.4 (5 μL)
#EP cut BAMT =
#EP cut BAMT = 13 ng/μL (4 μL)
#EP cut E0040 =
#EP cut E0040 = 11.7 ng/μL (5 μL)
#XP cut pSB1A3-1 =
#XP cut pSB1A3-1 = 22 ng/μL (2.5 μL)
#XP cut ATF1 =
#XP cut ATF1 = 28.1 ng/μL (2 μL)
#XP cut BSMT =
#XP cut BSMT = 15.7 ng/μL (3.5 μL)


'''We'll do 5 ligations, 10 μL total volume each:  
'''We'll do 5 ligations, 10 μL total volume each:  
Line 81: Line 81:


'''EP cut pSB1A3-1 and EP cut SAMT'''
'''EP cut pSB1A3-1 and EP cut SAMT'''
* μL water
*1.5μL water
*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells ''strongly'' like wet dog!!)
*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells ''strongly'' like wet dog!!)
*.5μL T4 DNA ligase enzyme
*.5μL T4 DNA ligase enzyme
*μL cut SAMT (50ng)
*5μL cut SAMT (50ng)
*μL cut backbone (50ng)
*2μL cut backbone (50ng)


'''EP cut pSB1A3-1 and EP cut BAMT'''
'''EP cut pSB1A3-1 and EP cut BAMT'''
*μL water
*2.5μL water
*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells ''strongly'' like wet dog!!)
*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells ''strongly'' like wet dog!!)
*.5μL T4 DNA ligase enzyme
*.5μL T4 DNA ligase enzyme
*μL cut BAMT (50ng)
*4μL cut BAMT (50ng)
*μL cut backbone (50ng)
*2μL cut backbone (50ng)


'''EP cut pSB1A3-1 and EP cut E0040 (+ Ctrl)'''
'''EP cut pSB1A3-1 and EP cut E0040 (+ Ctrl)'''
*μL water
*1.5μL water
*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells ''strongly'' like wet dog!!)
*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells ''strongly'' like wet dog!!)
*.5μL T4 DNA ligase enzyme
*.5μL T4 DNA ligase enzyme
*μL cut Barry's E0040 part (50ng)
*5μL cut Barry's E0040 part (50ng)
*μL cut backbone (50ng)
*2μL cut backbone (50ng)


'''XP cut pSB1A3-1 and XP cut ATF1'''
'''XP cut pSB1A3-1 and XP cut ATF1'''
*μL water
*4μL water
*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells ''strongly'' like wet dog!!)
*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells ''strongly'' like wet dog!!)
*.5μL T4 DNA ligase enzyme
*.5μL T4 DNA ligase enzyme
*μL cut ATF1 (50ng)
*2μL cut ATF1 (50ng)
*μL cut backbone (50ng)
*2.5μL cut backbone (50ng)
 
'''XP cut pSB1A3-1 and XP cut BSMT (+ Ctrl)'''
'''XP cut pSB1A3-1 and XP cut BSMT (+ Ctrl)'''
*μL water
*2.5μL water
*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells ''strongly'' like wet dog!!)
*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells ''strongly'' like wet dog!!)
*.5μL T4 DNA ligase enzyme
*.5μL T4 DNA ligase enzyme
*μL cut BSMT (50ng)
*3.5μL cut BSMT (50ng)
*μL cut backbone (50ng)
*2.5μL cut backbone (50ng)




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

Revision as of 11:38, 5 July 2006

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

    • 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

Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:MIT/2006/Notebook/2006-7-5&oldid=47070"