Haynes Lab:Notebook/Jan/2015/04/06: Difference between revisions

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(Autocreate 2015/04/06 Entry for Haynes_Lab:Notebook/Jan)
 
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==Entry title==
==04/06/15==
* Insert content here...
* Assembly - DPRE into "BD004"
* Sequencing - BD006/MV2
 
 
----
'''Assembly'''
* *'''[[User:Karmella Haynes|---Karmella]] 13:41, 6 April 2015 (EDT)''':
* BD004 appears to be missing the DPRE fragment. Gal4 sites and HPK are present (see alignment "BD004_P0001 (BD004_P0001.ab1)" on Benchling, https://benchling.com/s/0oSEQoHA/edit)
* BD006/MV2: insert BD006(E/S)/4182 + vector MV2(E/X)/4529
* Use Roche 2x buffer and NEB T4 ligase
* Concentrations of fragments: http://openwetware.org/wiki/Haynes_Lab:Notebook/Jan/2015/03/20
 
 
* Ligations
# 10 uL rxn, vector + insert
# 10 uL rxn, vector
# 15 uL rxn, vector + insert
# 15 uL rxn, vector
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | 1
| bgcolor=#cfcfcf | 2
| bgcolor=#cfcfcf | 3
| bgcolor=#cfcfcf | 4
|-
| DNA insert || 3.0 || --- || 5.5 || ---
|-
| DNA vector || 1.5 || 1.5 || 1.5 || 1.5
|-
| 2x buffer || 5.0 || 5.0 || 7.5 || 7.5
|-
| T4 ligase || 0.5 || 0.5 || 0.5 || 0.5
|-
| dH<sub>2</sub>O || --- || 3.0 || --- || 5.5
|-
| &nbsp; || 10 μL  || 10 μL  || 15 μL  || 15 μL
|}
 
--> RT/ ~30 min.
* Add 50 μL DH5-α turbo
* 5 min./ ice
* Plate on 100 ug/mL amp agar. Grow at 37 °C o/n
 
 
'''RESULTS''' (3/27/15)
# BD006/MV2, 10 uL rxn, vector + insert: 6 colonies
# BD006/MV2,10 uL rxn, vector: zero
# BD006/MV2,15 uL rxn, vector + insert: 4 colonies
# BD006/MV2,15 uL rxn, vector: zero
 
* Success! Pick two colonies from each plate (1 and 3) for minipreps
 
 
 
 




Revision as of 10:41, 6 April 2015

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04/06/15

  • Assembly - DPRE into "BD004"
  • Sequencing - BD006/MV2


Assembly


  • Ligations
  1. 10 uL rxn, vector + insert
  2. 10 uL rxn, vector
  3. 15 uL rxn, vector + insert
  4. 15 uL rxn, vector
Reagent 1 2 3 4
DNA insert 3.0 --- 5.5 ---
DNA vector 1.5 1.5 1.5 1.5
2x buffer 5.0 5.0 7.5 7.5
T4 ligase 0.5 0.5 0.5 0.5
dH2O --- 3.0 --- 5.5
  10 μL 10 μL 15 μL 15 μL

--> RT/ ~30 min.

  • Add 50 μL DH5-α turbo
  • 5 min./ ice
  • Plate on 100 ug/mL amp agar. Grow at 37 °C o/n


RESULTS (3/27/15)

  1. BD006/MV2, 10 uL rxn, vector + insert: 6 colonies
  2. BD006/MV2,10 uL rxn, vector: zero
  3. BD006/MV2,15 uL rxn, vector + insert: 4 colonies
  4. BD006/MV2,15 uL rxn, vector: zero
  • Success! Pick two colonies from each plate (1 and 3) for minipreps





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