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

Digest 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: ~2:50pm
Zymo
Eluted all samples with 10uL, except 7+11 and 9+20 (eluted with 20uL)
Ligate
Followed this protocol with the following amendments:
Set up 5 ligations (made MasterMix recipe x6)
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
  

Started Ligation at 4:15pm

Amy N. Kristofferson 18:37, 8 June 2010 (EDT)

To do:

• Analyze sequencing results from 7+10
• Miniprep iGEM10_001-3 samples.
• Grow up Lefty and Righty

Amy N. Kristofferson 13:20, 9 June 2010 (EDT)

Co-tranformation plate results for iGEM10_001-003

We plated the colonies we had picked to select for those containing iGEM10_001-003 on A,K and C in order to check for co-tranformation. Here are the results:

iGEM10_001a-d, iGEM10_002a-c, and iGEM10_003a were co-transformed.

Picking new iGEM10_001 colonies for colony PCR

Since iGEM10_001 didn't work at all (all colonies cotransformed), I picked 8 new colonies to perform colony pcr, inoculate and streak on triple antibiotic plates to check for cotransformation. Plates are in the incubator. Inoculations of colonies are upstairs in the warm room.

Miniprep of iGEM10_002d and iGEM10_003b,d

Miniprepped iGEM10_002d and iGEM10_003b,d to extract the plasmids. These colonies were not co-transformed, and 2d as welll as 3b looked successful on the colony pcr map. 3d didn't look as good on the map, but I miniprepped it anyway.

Map??? Sequence???

Colony PCR for iGEM10_001,8,9,014-016

I also performed the three steps outlines about for iGEM10_001,8,9,014-016. The plates and inoculations are stored in the same locations.

Here are the gel results:

Sequence Results from 7+10

7+10b and c sequenced perfectly, but 7c should be used since 7b showed signs of cotransformation (see sequence log for details)