User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/27: Difference between revisions

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** Total wells: 36 (1 well per rxn.)
** Total wells: 36 (1 well per rxn.)
** '''Template+water Multiplier''' = none (use 10x diluted stock for all rxns)
** '''Template+water Multiplier''' = none (use 10x diluted stock for all rxns)
** '''Primer+SYBR Mix Multiplier''' = 36 + 3 = '''39'''
** '''Primer+SYBR Mix Multiplier''' = 27 + 3 = '''39'''




Line 30: Line 30:
# Lu34_AA01 - 12
# Lu34_AA01 - 12
# Ga34_AA01 - 12
# Ga34_AA01 - 12
# nTc
# nTc (3 wells)




* Master Mix: Primers+SYBR
* Master Mix: Primers+SYBR
** 10.5 per well
{|
{|
|-
|-
|  Reagent || Volume || (x39)
|  Reagent || Volume || (x30)
|-
|-
| 750 nM F/R primers || 3.0 || 117.0
| 750 nM F/R primers || 3.0 || 90.0
|-
|-
| 2x SYBR MM || 7.5 || 292.5
| 2x SYBR MM || 7.5 || 225
|-
|-
|   || 15.0
|   || 15.0
|}
|}
* Plate loading
** Pipette 21 Primers+SYBR (2 x 10.5) into first row (A1-12)
** Multichannel-aliquot 10.5 Primers+SYBR into next row (B1-12)
** Pipette 10.5 into C1-3 (nTc wells)
** Multi-channel pipette 4.5 Template+water into appropriate wells
** Pipette 4.5 water into C1-3 (nTc wells)




Revision as of 17:31, 27 May 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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05/27/15

  • 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 = 27 + 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 (3 wells)


  • Master Mix: Primers+SYBR
    • 10.5 per well
Reagent Volume (x30)
750 nM F/R primers 3.0 90.0
2x SYBR MM 7.5 225
  15.0


  • Plate loading
    • Pipette 21 Primers+SYBR (2 x 10.5) into first row (A1-12)
    • Multichannel-aliquot 10.5 Primers+SYBR into next row (B1-12)
    • Pipette 10.5 into C1-3 (nTc wells)
    • Multi-channel pipette 4.5 Template+water into appropriate wells
    • Pipette 4.5 water into C1-3 (nTc wells)


  • 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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