Sbb14-Dingguo Chen

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Contents

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Team3: Organism: Thermotoga maritima Amino Acids: A,C,D,E,F,G,H,I,K,L

Protocols:

Design construction Files for All the Amino Acids.

Lab:

PCR(use expand polymerase) --> Run gel to check size of PCR parts --> Digestion Vector Digest Ligation(digest product + Vector + Ligase + ...)

--> Transformation --> Colonies selection and Screening.

Construction File

Protocols

Dingguo Chen 11:13, 4 March 2014 (PST)

PCR out the parts from the genome based on the designed construction files.

Dingguo Chen 12:20, 6 March 2014 (PST)

Run gel for soeing PCRs(Ala, Leu) and make sure they have the the correct size.

Run gel to check size of simple PCR products for other amino acides, then do zymo cleanup.

Results: All 4 soeing parts seem to have the correct sizes. for the normal PCRs, all but gul1 look good.

Dingguo Chen 11:45, 7 March 2014 (PST)

1. Redo PCRs for Glu1

2. Setting up Soeing PCRs.

Dingguo Chen 11:30, 11 March 2014 (PST)

Ran gel for PCR(Ala,and Leu). Results:

Image:Pcr3-11.jpg

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

Soeing PCR on Ala doesn't work and need to redo.

Dingguo Chen 11:53, 13 March 2014 (PST)

Run gel for Ala Soeing PCR-increase the concentration of the templates to 5 ul each this time.

Results: We got PCR part that have the desired size.


Plan for next lab: Do digestion and gel purification on all the PCR products.

Dingguo Chen 10:08, 18 March 2014 (PST)

Setting up digestion reactions for PCR products

Results:

Image:digest1.jpg Image:digest2.jpg


Task:

not finished. Digested products were melt in ADB buffered and stored for

next lab.

Dingguo Chen 10:48, 20 March 2014 (PST)

continue Digestion,

zymo clean the finished digested products.

Dingguo Chen 10:48, 1 April 2014 (PST)

ran an analytical gel to figure out vector and leu(messed up the label last time).

figured out which is which, and ready for ligation and transformation.

Dingguo Chen 10:57, 3 April 2014 (PST)

do ligation and transformation.

Dingguo Chen 10:39, 10 April 2014 (PST)

miniprep the culturs(44 samples)

Dingguo Chen 11:59, 24 April 2014 (PST)

Got back the two samples sent for sequencing, and they have perfect parts!

Today: Redo ligation and transformation for the other 9 digested samples

Result:

Image:4-24-gel.jpg

Lane1: ladder, Lane 2: Cys 1, Lane 3: Cys 2, Lane 4: Cys 4, Lane 5: Glu2 4, Lane 6: His 2, Lane 7: His 3, Lane 8: His 4, Lane 9: Ile 2, Lane 10: Ile 4

Dingguo Chen 11:23, 29 April 2014 (PST)

1. Picking colonies from transformed samples we got.

2. Run annalytical Gels for our samples on digest.

Dingguo Chen 11:10, 1 May 2014 (PST)

zymo cleaned our samples and digested our products

run gels

mapping for sequencing

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

Image:samplegel.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)

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