Berk2010-Amy: Difference between revisions

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


==[[User:Amy N. Kristofferson|Amy N. Kristofferson]] 13:20, 9 June 2010 (EDT)==
==[[User:Amy N. Kristofferson|Amy N. Kristofferson]] 13:20, 9 June 2010 (EDT)==

Revision as of 12:29, 9 June 2010

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.

Since iGEM10_001 didn't work at all (all colonies cotransformed), I will pick 8 new colonies to perform colony pcr, inoculate and streak to check for cotransformation.

I will miniprep 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'll miniprep it anyway.