User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/22: Difference between revisions

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==05/22/15==
==05/22/15==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Ryan - minipreps & digests
* Ryan - Bra minipreps & digests
* Cas-tone - minipreps & digests
* Cas-tone - minipreps & digests
* Ben - luc replacement donor plasmid
* Ben - luc replacement donor plasmid
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----
----
'''Minipreps'''<br>
'''Ryan - Bra minipreps & digests'''
* Check with EcoRI digests - promoter insertion generates a new EcoRI site
 
* Note: BraR = 776, pBra = 214
* Minipreps<br>
** Sigma GenElute kit, elute w/ 75 μL elution sln.
 
* Digests
** Check with EcoRI digests - promoter insertion generates a new EcoRI site
** Note: BraR = 776, pBra = 214


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->

Revision as of 11:05, 22 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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05/22/15

  • Ryan - Bra minipreps & digests
  • Cas-tone - minipreps & digests
  • Ben - luc replacement donor plasmid


Ryan - Bra minipreps & digests

  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  • Digests
    • Check with EcoRI digests - promoter insertion generates a new EcoRI site
    • Note: BraR = 776, pBra = 214
Reagent Volume Expected:
1-8. pBra-BraR_MRV = 2989, 1202
empty vector = 3977
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
dH2O 10.5
  15 μL --> 37°C/ ~15 min.


Assemblies

  1. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


  • Digests (Fermentas FD)
    • Specific notes
Reagent Volume
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight



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