Sbb14-GJordanRay

Graham Jordan Ray 15:56, 2 May 2014 (EDT)

Got sequencing data back.

Initial look showed messy reads for new parts. Possibly a result of a messy miniprep

Other reverse sequencing read looked good. Finished with 3 fully constructed parts.

Rest seemed to fail as a result of not clean vector digestions.

Graham Jordan Ray 12:58, 1 May 2014 (EDT)

Today we are miniing mapping and hopefully sending off for seqing

Our samples are Glu1 1-4, Phe, Gly, Ile, Cys

Glu1 1-4 cut with bsmbi: (3.2,2.3)

Ile cut with bsmbi: (4.4, 2.2)

Cys cut with bsmbi: (3.2,1.7,.6)

Phe cut with ncoi & hindIII: (4,3.3)

Gly cut with ncoi & hindIII: (4,2.9)

Vector cut with ncoi & hindIII: (4,1.3)

Map: Lane 1: ladder, lane 2: Glu1 1 (3.2,2.3), lane 3: Glu1 2 (3.2,2.3), lane 4: Glu1 3 (3.2,2.3), lane 5: Glu1 4 (3.2,2.3), lane 6: Ile (4.4,2.2) lane 7: Cys (3.2,1.7,.6), lane 8: Phe (4,3.3), lane 9: Gly (4,2.9), lane 10: Vector (4,1.3)

Graham Jordan Ray 13:06, 29 April 2014 (EDT)

The his sample we sent for sequencing looked perfect in the forward direction. Now we are requesting a reverse read to confirm the whole part.

Today we are rerunning the last of our old samples on digest to make sure none are the parts they are supposed to be

We also picked colonies from our limited transformation we had. Hopefully these all work well. We will mini map and seq on thursday

Gel maps:

1:

All are vector

Lane1: Ladder Lane2: Asp1 (3.2,2.6), Lane3: Asp2 (3.2,2.6), Lane4: Asp3 (3.2,2.6), Lane5: Ile1 (4.4,2.4), Lane6: Ile3(4.4,2.4), Lane7: Glu2 1(3.4,2), Lane8: Glu2 2(3.4,2), Lane9: Glu2 3(3.4,2), Lane10: Ladder

2:

All are vector

Lane1: Ladder Lane2:Glu1 1(3.2,2.3), Lane3:Glu1 2(3.2,2.3), Lane4:Glu1 3(3.2,2.3), Lane5:Glu1 4(3.2,2.3), Lane10: Ladder

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)

His looks good!

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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