User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/27

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05/27/15

  • Rene - hi-res melt curve screen on CRISPR clones
  • Ben - continue luc replacement donor cloning


Rene - hi-res melt curve screen on CRISPR clones


  • Set-up
    • 2 replicates per clone
    • Total wells: 36 (1 well per rxn.)
    • Template+water Multiplier = none (use 10x diluted stock for all rxns)
    • Primer+SYBR Mix Multiplier = 36 + 3 = 39


  • Template+ Water: Dilute PCR reaction 10x
    • Use multichannel to transfer 5.0 μL PCR products into 45.0 μL dH2O
    • Set up dilutions in 8-tube strips
    • Use 4.5 μL Template+water per rxn
  1. Lu34_AA01 - 12
  2. Ga34_AA01 - 12
  3. nTc


  • Master Mix: Primers+SYBR
Reagent Volume (x39)
750 nM F/R primers 3.0 117.0
2x SYBR MM 7.5 292.5
  15.0


  • Run PCR - Roche LC480
    • 95°C, 3 min
    • 30x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)]
    • 72°C, 30 sec
    • Melt: 70°C -- +0.1°C/ 5 sec (measure) --> 95°C





  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  • Digests
    • Check with #/# digests
Reagent Volume Expected:
1. BB 1 = size
2. BB2 = size
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Line item


  • 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
  1. insert/(a/b)/size + vector/(c/d)/size
  2. vector/(c/d)/size
Reagent Rxn1 Rxn2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL




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