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 50&mu;L
 * 3) the amount of dna desired is about 800 nanograms
 * 4) *BAMT pcr product at 58.8 ng/&mu;L -add 13.6 &mu;L
 * 5) 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:
 * 2) *28.9 &mu;L water
 * 3) *5 &mu;L NEB buffer 2
 * 4) *.5 &mu;L BSA
 * 5) *13.6 &mu;L BAMT pcr cleanup product (concentration = 50 ng/&mu;L)
 * 6) *.75 &mu;L Pst1
 * 7) *.75 &mu;L EcoR1
 * 8) *.5 &mu;L Dpn 1
 * 9) Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever.
 * 10) Did a PCR cleanup & nanodrop: concentration of digested BAMT = ??ng/&mu;L
 * 11) 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 50&mu;L
 * 3) the amount of dna desired is about 800 nanograms
 * 4) *ATF1 pcr product at 30.8 ng/&mu;L -add 26 &mu;L
 * 5) 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:
 * 2) *16.5 &mu;L water
 * 3) *5 &mu;L NEB buffer 3
 * 4) *.5 &mu;L BSA
 * 5) *26 &mu;L ATF1 pcr cleanup product (concentration = 50 ng/&mu;L)
 * 6) *.75 &mu;L Pst1
 * 7) *.75 &mu;L XbaI
 * 8) *.5 &mu;L Dpn 1
 * 9) Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever.
 * 10) Did a PCR cleanup & nanodrop: concentration of digested SAMT = ??ng/&mu;L
 * 11) run a gel before moving on to ligation !!

BACKBONE pSB1A3-1 bkb Xba/Pst Digest
BEGAN AT 11:15 AM
 * 1) *35 &mu;L water
 * 2) *5 &mu;L NEB buffer 3
 * 3) *.5 &mu;L BSA
 * 4) *8 &mu;L backbone plasmid
 * 5) *.75 &mu;L PstI
 * 6) *.75 &mu;L XbaI

CONTROL (BSMT-BB) Xba/Pst Digest

 * 1) *31.58 &mu;L water
 * 2) *5 &mu;L NEB buffer 3
 * 3) *.5 &mu;L BSA
 * 4) *11.42 &mu;L BSMT-BB (from BSMT miniprep @ 71ng/&mu;L )
 * 5) *.75 &mu;L PstI
 * 6) *.75 &mu;L 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: (?? &mu;L for 50 nanograms)
 * 1) EP cut pSB1A3-1 = 29.8 ng/&mu;L (2 &mu;L)
 * 2) EP cut SAMT = 10.4 (5 &mu;L)
 * 3) EP cut BAMT = 13 ng/&mu;L (4 &mu;L)
 * 4) EP cut E0040 = 11.7 ng/&mu;L (5 &mu;L)
 * 5) XP cut pSB1A3-1 = 22 ng/&mu;L (2.5 &mu;L)
 * 6) XP cut ATF1 = 28.1 ng/&mu;L (2 &mu;L)
 * 7) XP cut BSMT = 15.7 ng/&mu;L (3.5 &mu;L)

'''We'll do 5 ligations, 10 &mu;L 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
 * 1.5&mu;L water
 * 1&mu;L T4 DNA ligase buffer (vortex well before adding, make sure it smells strongly like wet dog!!)
 * .5&mu;L T4 DNA ligase enzyme
 * 5&mu;L cut SAMT (50ng)
 * 2&mu;L cut backbone (50ng)

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

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

XP cut pSB1A3-1 and XP cut ATF1
 * 4&mu;L water
 * 1&mu;L T4 DNA ligase buffer (vortex well before adding, make sure it smells strongly like wet dog!!)
 * .5&mu;L T4 DNA ligase enzyme
 * 2&mu;L cut ATF1 (50ng)
 * 2.5&mu;L cut backbone (50ng)

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

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