User:Karmella Haynes/Notebook/BioBrick cloning/2015/06/23: Difference between revisions

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* Digest & dephos inserts
* Digest & dephos inserts
** XbaI/NotI


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->

Revision as of 18:42, 23 June 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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06/23/15

  • Cas-tone - Assemblies: H3 parts into MV11
  • Rene - CRISPR library, continue dirty PCR


Cas-tone Assemblies

  • Do-over - use inserts with improved revrse primer (eliminate frame-shift)
  1. H3-1-40_MV11: H3-1-40/(X/N)/129 + MV11/(S/N)/9506
  2. H3-1-60_MV11: H3-1-60/(X/N)/189 + "
  3. H3-1-80_MV11: H3-1-80/(X/N)/250 + "
  4. H3-1-116_MV11: H3-1-116/(X/N)/373 + "
  5. H3-1-136_MV11: H3-1-136/(X/N)/433 + "


  • Digest of vector (Fermentas FD)
    • SpeI/NotI
Reagent Volume
DNA (plasmid) 20.0
10x buffer 3.0
SpeI 1.0
NotI 1.0
dH2O 5.0
  30 μL --> 37°C/ ~15 min.
  • Gel purification
    • Sigma GenElute kit. Elute & back elute w/ 25 μL elution sln.


  • DpnI digest & clean-up of PCR products
    • Add 1 μL DpnI to each PCR rxn. Incubate @ 37°C/ 5 min.
    • Use the Qiagen Qiaquick PCR Purification kit (cat. no. 28104 ) to purify DNA.
    • Elute & back-elute with 30 μL of elution solution.


  • Measure [DNA] for vector and inserts
Sample OD260 260/280 ng/μL
1. MV11 (S/N) 0.024 1.908 24.2
2. H3-1-40 PCR 0.024 1.908 24.2
3. H3-1-60 PCR 0.041 1.984 40.8
4. H3-1-80 PCR 0.044 1.917 43.8
5. H3-1-116 PCR 0.067 1.865 67.1
6. H3-1-136 PCR 0.072 1.901 72.4


  • Digest & dephos inserts
    • XbaI/NotI
Reagent Volume
DNA (500 ng) up to 15.0 μL
10X FD buffer 2.0
FD XbaI 1.0
FD NotI 1.0
Roche SAP 1.0
dH2O x μL
  20.0

Thermal cycler: LabNet OptiMax "AnOlig shrt"

  • 37°C, 10 min
  • 95°C, 5 min
  • Ramp down to 25°C, 5°C/1 min. [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min]
  • 25°C, ∞
  • Final concentration = 25 ng/μL.


  • Ligations
    • > 2:1 ratio calculations...
    • 433 bp largest insert / 9506 bp vector * 2 * 50 ng vector = 4.5 ng
    • 129 bp smallest insert / 9506 bp vector * 2 * 50 ng vector = 1.4 ng
Reagent Rxn1-5 Rxn6
Insert DNA 0.5 ---
Vector DNA 3.5 3.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 0.5
  10.0 μL 10.0 μL

RESULTS (5/22/15)

  • Success! Ligation plates have 15-20 colonies, neg. ctrl has 1 colony
  • Pick 2 colonies from each plate for 5 mL cultures and streaks



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