Team:CAU China/Experiments
Experiments & Protocols
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 pHSE400
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
The transformed strain was plated out on the solid LB medium which contained Ampicillin (A100)
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
Send the sample to the company for sequencing
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 plasmid to complete the experiment and then shift our genes to the pSB1C3 plasmid. We finish the shifting job while waiting.
Monday 20/4
PCR reaction on pVEL12C-EPC-A
Gel purification was performed on the PCR product
The product was named as A1508-(P)
Mix the A1508-(P) and pSB1C3-RpaR&I
Digest the mixture with restriction enzymes: XbaⅠ-HF and SpeⅠ
After four-hour digest, the product was purified with the silicagel column
Link for 4 hours under 16℃
The product was named as pSB1C3-A
Transform the Escherichia Coli (strain EP1300) with pSB1C3-A
The transformed strain was plated out on the solid LB medium which contained Ampicillin (A100)
Tuesday 21/4
There were many colonies growing up on the plate
Chose ten of them and scribed on another LB plate which contained Chloramphenicol (C25)
Wednesday 22/4
Colony PCR analysis
Chose two bacterial colonies from the agar plate from yesterday were transferred to 2 bottle of fluid LB medium to amplify bacteria
Thursday 23/4
Extract the pSB1C3-A plasmid from the bacteria
The plasmid was identified by enzyme digestion——XbaⅠ&SpeⅠ, NcoⅠ&StuⅠ
The clone was right
send the samples to the company for sequencing
Friday 24/4
The result of sequencing is right
Digest the pSB1C3-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 pSB1C3A-XS，BT/TB/BAG/GAB-XS
Link for 4 hours under 16℃
The products were named as pSB1C3-BAG/GAB/BAT/TAB
Transform the Escherichia Coli (strain EP1300) with pSB1C3A-XS，BT/TB/BAG/GAB-XS
The transformed strain was plated out on the solid LB medium which contained Chloramphenicol (C25)
Saturday 25/4
Choose about 10 of the grown-up colonies respectively and scribed on another LB plate which contained ampicillin (A100) for invert selection
chose the colonies which can grow on the Chloramphenicol plate but can’t grow on the ampicillin plate for colony PCR ananlysis
chose the right clone to send to the company for sequencing
Monday 27/4
The result of sequencing is right
Extract the four different plasmids and store
Wednesday 10/6
harvest the T0 generation seed
grow the seed on the MS mediun which contained hygromycin
Friday 19/6
chose the seeding which can grow on the antibiotic medium, and transferred them into the artificial soil