User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/20

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==mm/dd/yy==
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==03/20/13==
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* Line item 1
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* Oligo annealing, round 2
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* Line item 2
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* Assembly: annealed part into MV2
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----
----
'''Assemblies'''
'''Assemblies'''
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# MV9: 5' part/(a/b)/size + 3' part/(c/d)/size
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* New strategy for mammalian vector:  
 +
** Insert K-XbaI-NLS-6his-stop into the puro vector that has a deleted promoter. This way, the vector can be customized to carry different promoters.
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'''Oligo annealing'''
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* '''Oligo annealing'''
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# New BB 1
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* Redo oligo annealing with 99.9 and 97.9°C starting temperatures...
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{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
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* Previous Thermal cycling (3/19/13)
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| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
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** 99.9, 10 min.
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|-
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** [99.9, 1 min., -2°C; 90.0, 1 min., -2°C] x35
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| 10x annealing buffer || 2.0
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** 25°C, ∞
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|-
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** Labeled tubes A1, A2; Stored on bench o/n
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| dH<sub>2</sub>O || ---
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-
|-
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* New Thermal cycling (3/20/13)
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| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
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** 99.9, 10 min.
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|}
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** [99.9, 1 min., -2°C; 97.9, 1 min., -2°C] x36
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** 25°C, ∞
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** Labeled tubes A3, A4
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* Digests (Fermentas FD)
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* '''Digest''' (Fermentas FD)
** MV2, XbaI only
** MV2, XbaI only
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| dH<sub>2</sub>O || 5.0
| dH<sub>2</sub>O || 5.0
|-
|-
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
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| &nbsp; || 30 μL --> 37°C/ ~15 min.
|}
|}
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* Dephosphorylation (Roche)
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* '''Dephosphorylation''' (Roche)
{| {{table}} cellspacing="3" <!-- Dephos table -->
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
|-
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| 10x buffer d.p. || 2.0
| 10x buffer d.p. || 2.0
|-
|-
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| phosphatase || 1.0
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| rapid alk. phosphatase || 1.0
|-
|-
| dH<sub>2</sub>O || 10.8
| dH<sub>2</sub>O || 10.8
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{| {{table}} cellspacing="3" <!-- Ligations table -->
{| {{table}} cellspacing="3" <!-- Ligations table -->
|- bgcolor=#cfcfcf
|- bgcolor=#cfcfcf
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| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
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| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> 03/21/13</font>
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|-
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| 1. insert(Oligo S/S)/size, ## ng + MV2(X dp)/size, 25 ng || <font color="blue">MV9 #:1 (Pick #)</font>
 +
|-
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| 2. insert(Oligo S/S)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
|-
|-
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| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
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| 3. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
|-
|-
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| 2. vector(c/d)/ ## ng || &nbsp;
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| 4. vector(c/d)/ ## ng || &nbsp;
|}
|}

Revision as of 15:42, 20 March 2013

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

03/20/13

  • Oligo annealing, round 2
  • Assembly: annealed part into MV2



Assemblies

  • New strategy for mammalian vector:
    • Insert K-XbaI-NLS-6his-stop into the puro vector that has a deleted promoter. This way, the vector can be customized to carry different promoters.


  • Oligo annealing
  • Redo oligo annealing with 99.9 and 97.9°C starting temperatures...
  • Previous Thermal cycling (3/19/13)
    • 99.9, 10 min.
    • [99.9, 1 min., -2°C; 90.0, 1 min., -2°C] x35
    • 25°C, ∞
    • Labeled tubes A1, A2; Stored on bench o/n
  • New Thermal cycling (3/20/13)
    • 99.9, 10 min.
    • [99.9, 1 min., -2°C; 97.9, 1 min., -2°C] x36
    • 25°C, ∞
    • Labeled tubes A3, A4


  • Digest (Fermentas FD)
    • MV2, XbaI only
Reagent Volume
DNA (plasmid) 20.0 μL
10x buffer 3.0
XbaI 2.0
dH2O 5.0
  30 μL --> 37°C/ ~15 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. MV2 (XbaI) 0.081 1.841 81.3


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) 6.2 μL (500 ng)
10x buffer d.p. 2.0
rapid alk. phosphatase 1.0
dH2O 10.8
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) 03/21/13
1. insert(Oligo S/S)/size, ## ng + MV2(X dp)/size, 25 ng MV9 #:1 (Pick #)
2. insert(Oligo S/S)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
3. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
4. 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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