User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/22: Difference between revisions

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Dilute the purified dsDNA to 30 fmol/μL (30 nM)
Dilute the purified dsDNA to 30 fmol/μL (30 nM)
* Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
* Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
** Gal4DB-mCh (1170 bp) =
** ATF2 (906 bp) =
** MV10 (5191 bp) =
* Add x to dH<sub>2</sub>O, final vol. = 50 μL
* Add x to dH<sub>2</sub>O, final vol. = 50 μL
* Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction
* Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction
Prep the Oligo Bridges
* Note: Final conc. in LCR rxn. is 30 nM each
* Bring the IDT oligo pellet to 100μM with dH2O. nmoles oligo (on label) * 10 = μL H2O to add
* Make a 300 nM working solution (final volume = 100 μL) in a new tube. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL
LCR Reactions (PCR fragments / oligo bridges)
# Gal4DB-mCh, ATF2, MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc
# Neg: MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc
{| {{table}} cellspacing="3"
|- bgcolor=#cfcfcf
| Reagent || Rxn1 || Rxn2 (neg)
|-
| 30 nM DNA (3 nM) || 7.5 || 2.5
|-
| 300 nM Oligo Bridges (30 nM) || 7.5 || 7.5
|-
| 10X Ampligase Buffer || 2.5 || 2.5
|-
| Ampligase || 1.0 || 1.0
|-
| dH<sub>2</sub>O || 6.5 || 6.5
|-
| &nbsp; || 25.0
|}
Thermocycler: BioRad dual block
* 94 °C, 2 min.
* 50x[94°C, 10 sec; 55°C, 30 sec; 66°C, 60 sec
* 4 °C, ∞
Transformation
* Add to 60 μL DH5α-turbo; incubate 5 min/ice
* Plate on 100 μg/mL amp
* Grow at 37°C overnight




Revision as of 16:22, 22 April 2015

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04/22/15

  • Ryan - streak plates & liquid cultures (see 4/21/15)
  • Rene - minipreps: g53-2, g54
  • LCR Development - assembly attempt #1


LCR - assembly attempt #1


Column-purify PCR fragments

  • Use Qiagen PCR Clean-up, elute and back-elute w/ 30 μL elution buffer
  1. Gal4DB-mCh (4/22/15)
  2. ATF2 (4/22/15)
  3. MV10 (4/17/15)


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. 5'p Gal4DB-mCh PCR --- --- ---
2. 5'p ATF2 PCR --- --- ---
2. 5'p MV10 PCR --- --- ---


Dilute the purified dsDNA to 30 fmol/μL (30 nM)

  • Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
    • Gal4DB-mCh (1170 bp) =
    • ATF2 (906 bp) =
    • MV10 (5191 bp) =
  • Add x to dH2O, final vol. = 50 μL
  • Use 1.0 μL of diluted dsDNA per 10 μL LCR reaction


Prep the Oligo Bridges

  • Note: Final conc. in LCR rxn. is 30 nM each
  • Bring the IDT oligo pellet to 100μM with dH2O. nmoles oligo (on label) * 10 = μL H2O to add
  • Make a 300 nM working solution (final volume = 100 μL) in a new tube. 3 μL of 100μM oligo stock + 97 μL dH2O = 100 μL


LCR Reactions (PCR fragments / oligo bridges)

  1. Gal4DB-mCh, ATF2, MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc
  2. Neg: MV10 / LCRb_MV10_Gal4DB_rc, LCRb_mCh_ATF2_rc, LCRb_ATF2_MV10_rc
Reagent Rxn1 Rxn2 (neg)
30 nM DNA (3 nM) 7.5 2.5
300 nM Oligo Bridges (30 nM) 7.5 7.5
10X Ampligase Buffer 2.5 2.5
Ampligase 1.0 1.0
dH2O 6.5 6.5
  25.0

Thermocycler: BioRad dual block

  • 94 °C, 2 min.
  • 50x[94°C, 10 sec; 55°C, 30 sec; 66°C, 60 sec
  • 4 °C, ∞


Transformation

  • Add to 60 μL DH5α-turbo; incubate 5 min/ice
  • Plate on 100 μg/mL amp
  • Grow at 37°C overnight


Minipreps

  • Check with E/P 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.

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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