Berk2010-Conor: Difference between revisions

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*Transformed pBca1256-Bjh1934 into MG1655. Used procedure from (1)
*Transformed pBca1256-Bjh1934 into MG1655. Used procedure from (1)
*Attempted to enter parts into clotho --> would not save
*Attempted to enter parts into clotho --> would not save
===First step of Manual Assembly===
====Ligation:====
Followed [http://openwetware.org/wiki/Template:SBB-Protocols_Enz4 this protocol] with the following amendments: <br>
Mastermix
*6.5uL ddH2O
*1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
*0.5uL T4 DNA Ligase
DNA
*3uL Lefty vector
*3uL Right vector
Added DNA to MM at 5:12pm. Will incubate on bench until 5:42pm.
====Zymo====
Means of getting rid of restriction enzymes. Didn't heat kill because BamH1 isn't denatured by heat killing. Eluted with 10uL of H2O. Products are labeled 7+11 L, 9+8 L, 9+20 L.
====Digestion====
Lefty MM added to 9+8, 7+11, 9+20<br>
Right MM added to 7+23, 8+6, 7+5 <br>
They should be done incubating at 3:48pm.<br>
'''Lefty MasterMix''' (for 4uL miniprep DNA)
*4uL water
*1uL of NEB2
*0.5uL XhoI
*0.5uL BamH1
'''Righty MasterMix''' (for 4uL miniprep DNA)
*4uL water
*1uL of NEB2
*0.5uL XhoI
*0.5uL BglII
*note: righty digestions had different volumes, which seemed suspicious, so the digestion was repeated with 1uL of each enzyme for 30 min.


=To Do:=
=To Do:=

Revision as of 17:15, 7 June 2010

Conor McClune 14:38, 7 June 2010 (EDT)


  • (with Tahoura)
  • Transformed (spec)1256-2343 and (spec)1256-2344 into MG1655. Used minipreps of spec1256-2343 and spec1256-2344 from box and diluted 10x. Mixed 50ul MG1655 with 17.5ul KCN and added 1ul diluted DNA. Incubated for 1hr and plated on Spec. Left in incubator to grow
  • Plated pB6 (BAC), place in incubator. Needs to be picked and miniprepped tomorrow.
  • Transformed pBca1256-Bjh1934 into MG1655. Used procedure from (1)
  • Attempted to enter parts into clotho --> would not save

First step of Manual Assembly

Ligation:

Followed this protocol with the following amendments:
Mastermix

  • 6.5uL ddH2O
  • 1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
  • 0.5uL T4 DNA Ligase

DNA

  • 3uL Lefty vector
  • 3uL Right vector

Added DNA to MM at 5:12pm. Will incubate on bench until 5:42pm.

Zymo

Means of getting rid of restriction enzymes. Didn't heat kill because BamH1 isn't denatured by heat killing. Eluted with 10uL of H2O. Products are labeled 7+11 L, 9+8 L, 9+20 L.

Digestion

Lefty MM added to 9+8, 7+11, 9+20
Right MM added to 7+23, 8+6, 7+5
They should be done incubating at 3:48pm.


Lefty MasterMix (for 4uL miniprep DNA)

  • 4uL water
  • 1uL of NEB2
  • 0.5uL XhoI
  • 0.5uL BamH1

Righty MasterMix (for 4uL miniprep DNA)

  • 4uL water
  • 1uL of NEB2
  • 0.5uL XhoI
  • 0.5uL BglII
  • note: righty digestions had different volumes, which seemed suspicious, so the digestion was repeated with 1uL of each enzyme for 30 min.

To Do:

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