Ssb14-Jusuk Lee
13 Feb 2014
On Team 3:
Our amino acids are A,C,D,E,F,G,H,I,K,L
Our organism is Thermotoga maritima.
4 Mar 2014
We decided to synthesize everything together.
6 Mar 2014
Run gel for soeing PCRs(Ala, Leu).
Run gel to check size of simple PCR products for other amino acides, then do zymo cleanup.
All 4 SOEing parts look good and right sizes
lane 1: ala1, lane 2: ala2, lane 3: leu1, lane 4: leu2, lane 5: ladder
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)
7 Mar 2014
Reran PCR for Glu1(both at 2k45 one with and one without DMSO) Set up Soeing PCR
11 Mar 2014
Ran a gel on 2 SOEing PCRs and 2 re-trys of Glu PCRs
lane 1: ladder, lane 2: ala, lane 3: leu, lane 4: glu(1) +DMSO, lane 5: glu(1) normal
13 Mar 2014
Run gel for Ala Soeing PCR we increased the concentration of the templates to 5 ul.
We got PCR part that have the desired size.
18 Mar 2014
Setting up digestion reactions for PCR products
20 Mar 2014
zymo cleaned the finished digested product
3 Apr 2014
ligated our digested product with digested DNA.
then we transformed into cells and plated onto Amp plates
8 Apr 2014
picked 4 colonies on each plate and grew them in the media
10 Apr 2014
minipreped 44 samples
17 Apr 2014
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
Lys seems good
22 Apr 2014
Ran more samples on gel
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 seems good
24 Apr 2014
the result of Lys and Ala3 sequences were perfect. Religated and transformed the other 9 digested samples with newly digested vector Then ran another gel.
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)
Sent His for sequencing
29 Apr 2014
His was perfect in forward direction. Ran another set of gel but none of them looked good.
Then we picked colonies again and grew them under media
1 May 2014
zymo cleaned our samples and digested our products
Glu1 1-4,Ile, and Cys cut with bsmbi Phe,Gly, and Vector cut with ncoi & hindIII
Then we ran another gel
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)
2 May 2014
Got sequence results back Have three completely constructed parts: Lys, His, and Ala. Rest seems to have failed.
