Berk2010-Amy

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.

Amy N. Kristofferson 20:46, 7 June 2010 (EDT)

To do tomorrow:

• Colony PCR four colonies from each L/R plate. Make sure to innoculate each colony after and streak on triple antibiotic plates to check for co-transformation.
• Miniprep 7+10. Part #10 is 100bp, so we'll have to submit it for sequencing.
• Go through automated assemby excel and decide what strains to transform the basic parts into (Lefty or Righty?)
• Upload assembly tree pictures

Amy N. Kristofferson 17:15, 7 June 2010 (EDT)

First step of Manual Assembly: Creating iGEM10_003

Transformation

We Transformed 9+20,7+5 (iGEM10_003) and 9+8, 7+23 (iGEM10_001) into the Righty strain. And 7+11, 8+6 (iGEM10_002)into the Lefty strain.

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.