Berk2010-Conor: Difference between revisions
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===First step of Manual Assembly=== | ===First step of Manual Assembly=== | ||
====Digestion==== | ====Digestion==== | ||
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*note: righty digestions had different volumes, which seemed suspicious, so the digestion was repeated with 1uL of each enzyme for 30 min. | *note: righty digestions had different volumes, which seemed suspicious, so the digestion was repeated with 1uL of each enzyme for 30 min. | ||
====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. | |||
====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. | |||
=To Do:= | =To Do:= | ||
*miniprep BAC | |||
*pick stage 1 assembly | |||
Revision as of 17:23, 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
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.
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.
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.
To Do:
- miniprep BAC
- pick stage 1 assembly
