IGEM:MIT/2006/Notebook/2006-7-5: Difference between revisions
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*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''') | ||
General ligation considerations: 10μL total volume, about 50ng of each DNA | General ligation considerations: 10μL total volume, about 50ng of each DNA |
Revision as of 08:44, 5 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 !!
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 (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