Sbb14-GJordanRay

Graham Jordan Ray 15:12, 24 April 2014 (EDT)

The two sequencing runs we got back were both perfect!

Today we religated and transformed the other 9 digested samples into our new vector digest.

We also ran another get with a bsmbi digestion (vector will appear with one cut at 5.3:

Lane1: ladder, Lane 2: Cys 1: (3.2,1.7,.6), Lane 3: Cys 2: (3.2,1.7,.6), Lane 4: Cys 4: (3.2,1.7,.6), Lane 5: Glu2 4: (3.4,2), Lane 6: His 2: (2.7,2,.6), Lane 7: His 3: (2.7,2,.6), Lane 8: His 4: (2.7,2,.6), Lane 9: Ile 2: (4.4, 2.2), Lane 10: Ile 4: (4.4, 2.2)

Graham Jordan Ray 12:48, 22 April 2014 (EDT)

Looks like our vector is in all of our parts since our vector digest was not clean

As a result we are redoing a vector digest today. and running more samples

Lanes 1-3: Vector Lane 4: Ala2 (4,2.6), Lane 5: Ala3 (4,2.6), Lane 6: Glu2 2 (4,1.2,.2), Lane 7: Glu2 3 (4,1.2,.2), Lane8: Ile2 (4, 1.3, .9, .5),Lane9: Ile3 (4, 1.3, .9, .5), Lane 10: Ladder

Ala3 looks good

Graham Jordan Ray 14:37, 17 April 2014 (EDT)

Digested again:

Gel:

Lane1: Glu2 1 (4,1.2,.2), Lane2: Ala 1 (4,2.6), Lane3: Lys1 (4,1.5), Lane4: His1 (4,1.3), Lane5: Ile1 (4, 1.3, .9, .5), Lane6: Cys3 (4,1.4), Lane7: Cys4 (4,1.4), Lane8: Asp2 (4, 1.8), Lane9: Asp3 (4,1.8), Lane10: Ladder

Looks like Lys1 worked! give to chris for sequencing

Graham Jordan Ray 15:31, 15 April 2014 (EDT)

Digested minipreps with ncoI and hindIII

Messed up gels. Oh well.

Repeating.

Graham Jordan Ray 14:38, 10 April 2014 (EDT)

miniprepped all 44 liquid cultures

next time we will map and give Chris good samples for sequencing

Graham Jordan Ray 14:45, 8 April 2014 (EDT)

pick 4 colonies from each plate in to media with amp in it

Counts of cells:

Gly: 90

His: 100

Lys: 100

Glu1: 70

Glu2: 60

Ile: 40

Ala: 40

Asp: 35

Cys: 30

Phe: 50

Leu: 70

forgot to do control

Graham Jordan Ray 13:02, 3 April 2014 (EDT)

Today we ligated all 11 of our digests with digested DNA.

After we transformed into cells and plated onto Amp plates

Graham Jordan Ray 12:46, 1 April 2014 (EDT)

Digested more template on 3/20:

Graham Jordan Ray 15:13, 18 March 2014 (EDT)

Ran a digest on all samples according to map below and ran a gel.

lane 1:ladder, lane2:glu2 (.6&.8 parts), lane3:Ile(2.8) lane 4: leu (2.5) lane 5: vector (4) lane 6: Cys (1.4) lane 7: Asp (1.8) lane 8: Glu (1.5) lane 9 phe (3.4) lane 10: Gly (2.9)

Graham Jordan Ray 13:43, 13 March 2014 (EDT)

Ran a gel on Ala SOEing PCR- added more of tempates this time (5 uL of each)

lane 1: ladder, lane 2: ala, lane 3: other groups sample

Zymo'd Ala sample

Next tuesday we will digest, and gel purify. If time we can ligate and transform!

Graham Jordan Ray 13:09, 11 March 2014 (EDT)

Ran a gel on 2 SOEing PCRs and 2 re-trys of Glu PCRs ( both at 2k45 one with and one without DMSO)

lane 1: ladder, lane 2: ala, lane 3: leu, lane 4: glu(1) +DMSO, lane 5: glu(1) normal

Did Zymos on 3 parts that worked. Now have 10/11 of our parts

Reran SOEing PCR on Ala

Graham Jordan Ray 15:14, 7 March 2014 (EST)

Plan for today:

Look at images of gels all looked good except glu1

Repeating Glu1, failed 1st time

Set up SOEing PCRs.

Digest vector and parts, run on gel, and then do a gel purification on tuesday

NcoI/HindIII (Buffer 2):

• Vector

• Cys

• Asp

• Glu

• Phe

• Gly

• His

• Lys

• Ala

BsaI/HindIII (Buffer 3):

• Ile

• Leu

BsmbI/HindIII (Buffer 3):

• Glu(2)

Graham Jordan Ray 13:10, 6 March 2014 (EST)

Plan for today:

Run gel on pre-soeing PCRs (ALA and LEU) & make sure they are the correct size.

If they are the correct sizes gel purify, and run SOEing PCR.

Concurrently for simple PCRs we are running a gel to check sizes and then we will zymo PCRs to cleanup.

Results of todays Gels:

All 4 SOEing parts look good and right sizes:

lane 1: ala1, lane 2: ala2, lane 3: leu1, lane 4: leu2, lane 5: ladder

All but 1 of normal PCRs look good:

lane 1: ladder, lane 2: cys (1.4), lane 3: asp (1.7) lane 4: glu (1.5) lane 5: phe (3.4) lane 6: gly (2.9) lane 7: his (1.2) lane 8: ile (2.8) lane 9 lys (1.5) lane 10:Glu(2) (1.4)

tutorials

Graham Jordan Ray 12:56, 4 March 2014 (EST)

Today we started assembly of our parts.

We PCRd on all of our parts off of the genome based on the design file

Graham Jordan Ray 13:16, 18 February 2014 (EST)

our design spreadsheet: Group 3 desing

Graham Jordan Ray 13:16, 13 February 2014 (EST)

On Team 3:

Our amino acids are A,C,D,E,F,G,H,I,K,L

Our organism is Thermotoga maritima.

Found at ATCC Link

Genomic DNA found at Thermotoga maratima

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