Berk2010-Amy
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.
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 17:15, 7 June 2010 (EDT)
First step of Manual Assembly of iGEM10_007: Creating iGEM10_001-3
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
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 5:17pm. Will incubate on bench until 5:37pm.
Transformation
We Transformed 9+20,7+5 (iGEM10_003) and 9+8, 7+23 (iGEM10_001) into the Lefty strain. And 7+11, 8+6 (iGEM10_002)into the Righty strain.
Amy N. Kristofferson 20:46, 7 June 2010 (EDT)
To do tomorrow:
- Go through automated assembly excel and decide what strains to transform the basic parts into (Lefty or Righty?)
Amy N. Kristofferson 13:18, 8 June 2010 (EDT)
Miniprep and Sequencing Submission for 7+10b,c
Miniprepped 7+10b and c. Put 8uL of each in Eppendorph tubes and submitted for sequencing. Skipped mapping step because part is only 100bp.
Colony PCR of iGEM10_001-003
Selected four colonies from each plate. Dipped the pipette tip in PCR tube with 40uL of MasterMix and a well containing 3mL of LB with the appropriate antibiotics, before spreading the pipette tip across a plate with all three antibiotics (to check for cotransformation).
iGEM10_001 (part 8 next to 23 in a CK vector): Expected band length: about 1500bp
iGEM10_002 (part 11 next to 6 in AC vector): Expected band length: about 550bp
iGEM10_003 (part 20 next to 5 in CK vector): Expected band length: about 1250bp
Colony PCR
Lane 1 iGEM10_003a (should be 1250bp)
Lane 2 iGEM10_003b (should be 1250bp)
Lane 3 iGEM10_003c (should be 1250bp)
4 iGEM10_003d (should be 1250bp)
5 iGEM10_001a (should be 1500bp)
6 iGEM10_001b (should be 1500bp)
7 iGEM10_001c (should be 1500bp)
8 iGEM10_001d (should be 1500bp)
9 iGEM10_002a (should be 550bp)
10 iGEM10_002b (should be 550bp)
11 iGEM10_002c (should be 550bp)
12 iGEM10_002d (should be 550bp)
We will choose the following samples to miniprep tomorrow:
iGEM10_001c,d
iGEM10_002c,d
iGEM10_003a,b
First step of Manual Assembly of iGEM10_013 and 20: Creating iGEM10_008-009 and 014-016
Digested 8+4, 7+11 (x2), 9+20 (x2) with Lefty MasterMix.
Digested 9+3, 8+1, 7+2, 7+19, 8+12 with Righty MasterMix.
Start time: 1:50pm Taken out of incubator at:
