Team:SYSU-CHINA/Labjournals
1. Transformation and plate spreading
Three bottles of plasmid dry powder (pCMV-tag 3b, pGPU6-GFP-Neo, pTet-on-Advanced) were dissolved in UD water and transfected into competent Escherichia coli. Spread the three transformed bacteria on LB plate with Kan, 37 ° C overnight.
1. Inoculation
A single colony of the three kinds of bacteria spread on the plate on September 26 was selected and amplified with LB medium (containing antibiotics).
1. Extraction of plasmid
Plasmid pCMV-Tag 3B, pGPU6-GFP-Neo, pTet-On-Advanced were extracted using TIANprep Mini Plasmid Kit.
2. DNA concentration detection:
3. PCR amplification
1)Target: apoptin
Template: P2A-STEM-positive-apoptin
Primer: apoptin-F/R
Enzyme: Phusion
2) Target: Ribo_ligation-dsRNA
Template: Ribo_ligation-dsRNA
Primer: cyclic-dsRNA-F/R
Enzyme: Phusion
3) Identification by agarose electrophoresis: product positive
4) Gel extraction: apoptin and Ribo_ligation-dsRNA were extracted using ZymocleanTM Gel DNA Recovery Kit.
Concentration detection: apoptin = 28.8ng/μl，dsRNA = 23.9 ng/μl
4. Transformation and plate spreading:
Plasmid pTRE2hyg was transfected into E.coli, and spread on LB plate with Amp, 37°C overnight.
5. Breed conservation:
30μl bacterial solution + 70μl glycerinum, conserved at -80°C
1. Double enzyme digestion:
pCMV digested with two groups of enzymes (①NotI-HF & EcoRV；②NheI-HF & EcoRV),magnetic beads recycling.
pGPU6 digested with EcoRI-HF & NotI-HF, magnetic beads recycling.
2. PCR amplification:
1)Target: GFP ①
Template: pGPU6-GFP-Neo
Primer: GFP-F1/R1
Concentration: 828.3ng/μl
Target: GFP ②
Template: pGPU6-GFP-Neo
Primer: GFP-F2/R1
Concentration: 779.5ng/μl
Target: GFP ③
Template: pGPU6-GFP-Neo
Primer: GFP-F3/R3
Concentration: 543.7ng/μl
2) Target: P2A-STEM-apoptin（experimental）①
Template: P2A-STEM-apoptin
Primer: STEM-apoptin-F1/R1
Concentration: 530.4ng/μl
Target: P2A-STEM_positive-apoptin（positive control）①
Template: P2A-STEM-apoptin
Primer: STEM-apoptin-F1/R1
Concentration: 521.7ng/μl
Target: P2A-STEM_negative-apoptin（negetive control）①
Template: P2A-STEM-apoptin
Primer: STEM-apoptin-F1/R1
Concentration: 502.9ng/μl
Target: P2A-STEM_negative-apoptin（experimental）②
Template: P2A-STEM-apoptin
Primer: STEM-apoptin-F1/R2
Concentration: 508.7ng/μl
3. Inoculation
A single colony of the three kinds of bacteria spread on the plate on September 28 was selected and amplified with LB medium with Amp， 37°C overnight.
1. Extraction of plasmid
Plasmid pTRE2hyg was extracted using TIANprep Mini Plasmid Kit.
Double enzyme digestion:
pCMV digested with two groups of enzymes(①NotI-HF & EcoRV；②NheI-HF & EcoRV), magnetic beads recycling.
pTRE digested with two groups of enzymes①NheI-HF、SalⅠ；②NheI-HF、EcoRV), magnetic beads recycling;
pGPU6 digested withEcoRI-HF、NotI-HF, magnetic beads recycling;
2. Detection of concentration:
3. Identification by agarose electrophoresis.
Identify PCR product on September 29. Only 3 groups of GFP were successfully recombined. 4. Extract GFP using ZymocleanTM Gel DNA Recovery Kit.
4. Extract GFP using ZymocleanTM Gel DNA Recovery Kit.
Concentration:
1. PCR recombination
1) Vector: pCMV insert element: apoptin
2) Vector: pGPU6 insert element: Ribo_ligation-dsRNA
2.PCR
GFP: sample lost, PCR one more time. product positive.
P2A-STEM-apoptin：product negative，target was about 500bp，product was about 1000bp. Suspect that UD water was contaminated or annealing temperature was wrong. re-PCR as follow:
Investigate the influence of water:
Two experimental groups:
P2A-STEM-apoptin 1μl（5ng）
STEM-apoptin-F1 1.25μl
STEM-apoptin-R1/R2 1.25μl
phusion 12.5μl
ddH2O 9μl
Total 25μl
Two negative control:
STEM-apoptin-F1 1.25μl
STEM-apoptin-R1/R2 1.25μl
phusion 12.5μl
ddH2O 10μl
Total 25μl
(2) Investigate the influence of temperature(low down annealing temperature from 60°C to 55°C):
Target：P2A-STEM-apoptin ①（520bp）
Primer：STEM-apoptin-F1/R1
Target：P2A-STEM_positive-apoptin ①（520bp）
Primer：STEM-apoptin-F1/R1
Target：P2A-STEM_negative-apoptin ①（502bp）
Primer：STEM-apoptin-F1/R1
Target：P2A-STEM-apoptin ②（520bp）
Primer：STEM-apoptin-F1/R2
And two negative control without template
3. Gel extraction:
The electrophoretic results were still abnormal, and sequencing was needed to compare the results.
4. Transformation and plate spreading:
plasmid pCMV-apoptin was transfected into E.coli(experimental group, positive control, negative control, respectively)
Plasmid pGPU6-Ribo-ligation-dsRNA was transfected into E.coli(experimental group, positive control, negative control, respectively)
Spread on LB plate, 37°C overnight.
1. Colony PCR:
Use 10.1 plate, pick 8 pCMV-apoptin and pGPU6-Ribo-ligation-dsRNA positive
colonies(experimental group) respectively for colony PCR amplification.
identify by agarose electrophoresis, choose 3 groups of positive product and amplify with LB medium.
2. PCR recombination
Vector: pTRE
Insert element: GFP
3. Transformation and plate spreading
Plasmid pTRE-GFP was transfected into E.coli, and spread on LB plate with Amp, 37°C overnight.
1. Colony PCR:
Use 10.2 plate, pick 8 pTRE-GFP positive colonies for colony PCR amplification.
identify by agarose electrophoresis, choose 2 groups of positive product and amplify them with LB medium.
2. Inoculation
Antibiotic added on 10.2 was wrong, re-inoculate today
Plasmid: pCMV-apoptin and pGPU6-Ribo-ligation-dsRNA
1. Subculturing HEK293 cells:
Tissue: HEK293 cells
Reagents: serum, trypsin, PBS buffer, DEME medium
The following is the detailed protocol of this experiment, the subsequent subculture of cells will not be described too much.
1. Subculturing cells:
13:00 Transfer HEK293 to a 6-well plate (2 wells) and place it in a 37°C incubator.
1. Subculturing cells:
16:00 Transfer HEK293 to a 6-well plate (2 wells) and place it in a 37°C incubator.
1. Subculturing cells:
15:00 Transfer the new HEK293 to the 6-well plate and place it in the 37°C incubator.
1. Cell transfection:
A total of 6 groups were transfected: ①pCMV-apoptin 1-1-4; ②pCMV empty vector; ③pGPU6-dsRNA 2-1-5; ④pGPU6 empty vector; ⑤pTRE-GFP 3-1-1&pTet-on; ⑥pTRE empty vector &pTet-on.
Note: 1. Only the plasmid, G418 and hygromycin were transferred; 2. The amount of plasmid is 2ug, which should be taken according to the concentration of the plasmid solution; 3. The transfected cells are at the bottom of the upper incubator Cu ltivate, with words such as "iGEM, HEK293, No. 1~6" written.
1. Observe the transfected HEK293 cells and subculturing HEK293.
1. Cell observation:
Observe several wells labeled pCMV-apoptin, pCMV, pGPU6-ribo-ligation-dsRNA, pGPU6.
2. DOX induction:
The four experimental groups pTRE-GFP+pTet-on and pTRE-GFP-STEM-apoptin+pTet-on each add about 700μL of medium containing 0/0.1/1/2μg/mLDOX, and place them in the incubator. subculturing:
Transfer to a 6-well plate with 2 wells, a 24-well plate with 10 wells, mark "iGEM 10.20", and place in the incubator.
1. Cell observation:
Observed the growth and green fluorescence of GFP-STEM-apoptin, pTRE-GFP+pTet-on, and Dox concentration of 0, 0.1, 1, 2.
2. RNA extraction:
Use ZYMO RESEARCH Direct-zolTM RNA MiniPrep kit to extract RNA.
Obtained concentration: control group: 67ng/μL; experimental group: 40.9ng/μL.
1. Cell transfection:
pCMV empty plasmid control
pCMV-STEM-apoptin
pCMV-STEM_negative
pCMV-STEM_positive
A total of four groups.
1. Observe the four groups of cells transfected with October.22.
2. Subculturing: Pass 6-well plate with 6 holes.
Address:
No.135,Xingang Xi Road,
Guangzhou,510275,P.R.China
Tel:+86-20-84112828
