Berk2010-Amy
Amy N. Kristofferson 17:15, 7 June 2010 (EDT)
First step of Manual Assembly: Creating iGEM10_003
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 5:17pm. Will incubate on bench until 5:37pm.
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
Amy N. Kristofferson 16:56, 7 June 2010 (EDT)
Colony PCR of 7+10 (we forgot to do this last week).
Lane 1: ladder
Lane 2: sample a
Lane 3: b
Lane 4: c
Lane 5: d
Analysis: Part is about 100bp, so all samples look correct, because the PCR product should be around 300bp.
To do: Allow inoculations to grow to saturation overnight. Miniprep to extract plasmids tomorrow.
Amy N. Kristofferson 14:24, 7 June 2010 (EDT)
Colony PCR of 7+10
(vector=7, part=10)
Ran Col500PCR on four colonies picked off of 7+10 plate. Also innoculated each colony in 3mL of LB-KA. Put tubes in shaker upstairs.
