Ssb14-Jusuk Lee

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Contents

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.

Construction File


Protocols

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

Image:Pcr3-11.jpg

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 Image:digest1.jpg Image:digest2.jpg

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:

Image:pic4172014.jpg

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 Image:group3pic42214.jpg

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.

Image:Gel42414.jpg

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


Image:Gel5-1-14.jpg

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.

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