User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/15: Difference between revisions

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'''Assemblies'''
'''Assemblies'''
# pAub-AubR_MRV: pAub-PCR(E/S dp) + AubR/MRV(BbsI)  
* For Ryan and Ben
# pBja-BjaR_MRV: pBja-PCR(E/S dp) + BjaR/MRV(BbsI)
# pAub-AubR_MRV: pAub-PCR(E/S dp)/153 + '''AubR/MRV(BbsI)/4087'''
# pBra-BraR_MRV: pBra-PCR(E/S dp) + BraR/MRV(BbsI)
# pBja-BjaR_MRV: pBja-PCR(E/S dp) + '''BjaR/MRV(BbsI)/3977'''
# pRpa-RpaR_MRV: pRpa-PCR(E/S dp) + RpaR/MRV(BbsI)
# pBra-BraR_MRV: pBra-PCR(E/S dp) + '''BraR/MRV(BbsI)/3977'''
# DBN007_pSB1A3: DBN006(BsaI) 1972 + DBN001(BsaI) 1267/1069(2336)
# pRpa-RpaR_MRV: pRpa-PCR(E/S dp) + '''RpaR/MRV(BbsI)/3980'''
# DBN007_pSB1A3: '''DBN006(BsaI)/1972''' + '''DBN001(BsaI)/1267/1069(2336)'''




* Digests (Fermentas FD)
# AubR/MRV(BbsI)/4087
# BjaR/MRV(BbsI)/3977
# BraR/MRV(BbsI)/3977
# RpaR/MRV(BbsI)/3980
# DBN006(BsaI)/1972
# DBN001(BsaI)/1267/1069(2336) (two bands)


* Digests (Fermentas FD)
** Specific notes


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

Revision as of 11:10, 15 May 2015

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mm/dd/yy

  • Ryan - assembly (traditional) for Receiver plasmids
  • Ben - assembly for luc replacement


Assemblies

  • For Ryan and Ben
  1. pAub-AubR_MRV: pAub-PCR(E/S dp)/153 + AubR/MRV(BbsI)/4087
  2. pBja-BjaR_MRV: pBja-PCR(E/S dp) + BjaR/MRV(BbsI)/3977
  3. pBra-BraR_MRV: pBra-PCR(E/S dp) + BraR/MRV(BbsI)/3977
  4. pRpa-RpaR_MRV: pRpa-PCR(E/S dp) + RpaR/MRV(BbsI)/3980
  5. DBN007_pSB1A3: DBN006(BsaI)/1972 + DBN001(BsaI)/1267/1069(2336)


  • Digests (Fermentas FD)
  1. AubR/MRV(BbsI)/4087
  2. BjaR/MRV(BbsI)/3977
  3. BraR/MRV(BbsI)/3977
  4. RpaR/MRV(BbsI)/3980
  5. DBN006(BsaI)/1972
  6. DBN001(BsaI)/1267/1069(2336) (two bands)


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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