Difference between revisions of "Team:CAU China/Notebook"

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Notebook

Lab Journal

"Do not, for one repulse, forgot the purpose that you resolved to effort." - William Shakespeare

Wednesday 1/4

We started our work in the wet lab with a one-day lab safety course. We learned basic lab safety knowledge, such as how to put out a fire in the lab, how to deal with the poisonous chemical and the dangerous bacteria.

Thursday 2/4

We started to get familiar with our lab. We learned about where we can find everything that is needed for working in the lab. In addition, we had a lab meeting about the project goals and the way how to get there.

Friday 3/4

PCR reaction on pER41G-A

Gel purification was performed on the PCR product

The product was named as Ap-PCR(P)

Mix the Ap-PCR(P) and pER41G-A

Digest the mixture with restriction enzymes: XbaⅠ-HF and SacⅠ

After four-hour digest, the product was purified with the silicagel column

Linkage

Link overnight

The product was named as pHSE-A

Saturday 4/4

Transform the Escherichia Coli (strain EP1300) with pHSE-A

Mix EP1300 and pHSE-A, 0℃ice bath 30min

42℃ water bath 90s

0℃ice bath 1min

37℃recover 1h

Sunday 5/4

There were many colonies growing up on the plate

Chose ten of them and scribed on another LB plate which contained kanamycin (K100)

Monday 6/4

Three bacterial colonies from the agar plate from yesterday were transferred to 3 bottle of fluid LB medium to amplify bacteria

Extract the pHSE-A plasmid from the bacteria

The plasmid was identified by enzyme digestion——XbaⅠ&SacⅠ, NcoⅠ&StuⅠ

The clone was right

Tuesday 7/4

Digest the pHSE-A and pUC57-BT/TB/BAG/GAB respectively with XbaⅠ-HF and StuⅠ

After four-hour digest, the products were then analysed on gel-electrophoresis and purified with the silicagel column

The products were named as pHSE-A-XS and BT/TB/BAG/GAB-XS

Linkage

Link overnight

The products were named as pHSE-BT/TB/BAG/GAB

Wednesday 8/4

Transform the Escherichia Coli (strain EP1300) with pHSE-BT/TB/BAG/GAB

The transformed strain was plated out on the solid LB medium which contained kanamycin (K100)

Thursday 9/4

Choose about 10 of the grown-up colonies respectively and scribed on another LB plate which contained ampicillin (A100) for invert selection

Friday 10/4

Choose the colonies which can grow on the kanamycin plate but can’t grow on the ampicillin plate

Colony PCR analysis

Two bacterial colonies from each agar plate were transferred to 8 bottle of fluid LB medium to amplify bacteria.

Extract the plasmid from the bacteria

The plasmid was identified by enzyme digestion——XbaⅠ-HF and StuⅠ

Monday 13/4

The result of sequencing is right

Transform the Agrobacterium tumefaciens(GV3101) with pHSE-BT/TB/BAG/GAB

The transformed strain was plated out on the solid LB medium which contained Gentamicin, rifampicin and kanamycin

Tuesday 14/4

Choose 3 of the grown-up colonies in each plate for colony PCR analysis

Choose one of each and transferred to 4 bottle of fluid LB medium to amplify bacteria.

Wednesday 15/4

Let the transformed Agrobacterium tumefaciens(GV3101) infect the wild type arabidopsis thaliana

16/4-10/6

waiting for seed bearing of arabidopsis thaliana. Because the standard biobirck plasmid is not a binary vector, we have to use our own plaamid to complete the experiment and then shift our genes to the pSB1C3 plasmid. We finish the shifting job while waiting.