IGEM:MIT/2006/Notebook/2006-7-5: Difference between revisions
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#Did a PCR cleanup & nanodrop: concentration of digested BAMT = ??ng/μL | #Did a PCR cleanup & nanodrop: concentration of digested BAMT = ??ng/μL | ||
#'''run a gel before moving on to ligation !!''' | #'''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== | ==ATF1 PCR Product Xba/Pst Digest== | ||
Line 48: | Line 86: | ||
#*.75 μL XbaI | #*.75 μL XbaI | ||
==CONTROL ( | ==CONTROL (BSMT-BB) Xba/Pst Digest== | ||
#* | #*31.58 μL water | ||
#*5 μL NEB buffer | #*5 μL NEB buffer '''3''' | ||
#*.5 μL BSA | #*.5 μL BSA | ||
#* | #*11.42 μL BSMT-BB (from BSMT miniprep @ 71ng/μL ) | ||
#*.75 μL | #*.75 μL PstI | ||
#*.75 μL | #*.75 μL XbaI | ||
==Glycerols and Miniprep of BSMT biobrick== | |||
#Did minipreps of A, C, E BSMT liquid cultures to get the BSMT biobrick plasmid. | |||
#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) | ||
#EP cut pSB1A3-1 = 29.8 ng/μL (2 μL) | |||
#EP cut SAMT = 10.4 (5 μL) | |||
#EP cut BAMT = 13 ng/μL (4 μL) | |||
#EP cut E0040 = 11.7 ng/μL (5 μL) | |||
#XP cut pSB1A3-1 = 22 ng/μL (2.5 μL) | |||
#XP cut ATF1 = 28.1 ng/μL (2 μL) | |||
#XP cut BSMT = 15.7 ng/μL (3.5 μL) | |||
'''We'll do 5 ligations: | '''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 SAMT | ||
*EP cut pSB1A3-1 and EP cut BAMT | *EP cut pSB1A3-1 and EP cut BAMT | ||
*EP cut pSB1A3-1 and EP cut E0040 ('''+ Ctrl''') | *EP cut pSB1A3-1 and EP cut E0040 ('''+ Ctrl''') | ||
*XP cut pSB1A3-1 and XP cut ATF1 | *XP cut pSB1A3-1 and XP cut ATF1 | ||
*XP cut pSB1A3-1 and XP cut | *XP cut pSB1A3-1 and XP cut BSMT ('''+ Ctrl''') | ||
''' | '''EP cut pSB1A3-1 and EP cut SAMT''' | ||
*1.5μL 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 | ||
*5μL cut | *5μL cut SAMT (50ng) | ||
*2μL cut backbone (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 | *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 | ||
*5μL cut Barry's | *5μL cut Barry's E0040 part (50ng) | ||
*2μL cut backbone (50ng) | *2μL cut backbone (50ng) | ||
Ligate at room temp for 15 to 20 minutes and then proceed to transformation | '''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''' |
Latest revision as of 08:32, 6 July 2006
BAMT PCR Product Eco/Pst Digest
BEGAN AT 11:15 AM
- We are cutting the BAMT pcr product (using EcoRI and PstI)--
- Total reaction volume is 50μL
- the amount of dna desired is about 800 nanograms
- BAMT pcr product at 58.8 ng/μL -----add 13.6 μL
- 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)
- 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
- Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever.
- Did a PCR cleanup & nanodrop: concentration of digested BAMT = ??ng/μL
- 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
- We are cutting the ATF1 pcr product (using XbaI and PstI)--
- Total reaction volume is 50μL
- the amount of dna desired is about 800 nanograms
- ATF1 pcr product at 30.8 ng/μL -----add 26 μL
- 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)
- 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
- Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever.
- Did a PCR cleanup & nanodrop: concentration of digested SAMT = ??ng/μL
- 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
- Did minipreps of A, C, E BSMT liquid cultures to get the BSMT biobrick plasmid.
- 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)
- EP cut pSB1A3-1 = 29.8 ng/μL (2 μL)
- EP cut SAMT = 10.4 (5 μL)
- EP cut BAMT = 13 ng/μL (4 μL)
- EP cut E0040 = 11.7 ng/μL (5 μL)
- XP cut pSB1A3-1 = 22 ng/μL (2.5 μL)
- XP cut ATF1 = 28.1 ng/μL (2 μL)
- 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