Difference between revisions of "Team:ECUST/Experiment"

Materials:

5xl Gxl Buffer
Primer star Gxl（DNA polymerase）
dNTP
ddH₂O
Rhodobacter sphaeroides 2.4.1 genome
plasmid backbone with sYFP2 gene

Forward/Reverse Primer：

Method:

PCR program:

PCR System（50ul）：

Materials:

PCR fragment (concentration requirement> 100 ng / ul)
Linearized plasmids (concentration requirements> 100 ng / ul, can be prepared by digestion or reverse PCR)
1.33x ITA reagent

Method:

1. Add all kinds of PCR fragments and linear plasmids of the same mass to 1.33x ITA reagents (7.5 µL), making total volume 10 µL
2. Keep the system at 50°C for 1h
3. Transform 10 µL ligation liquid and select using chl resistance

Materials:

10xl Taq Buffer
Taq polymerase
dNTP
ddH₂O

Forward/Reverse Primer：

Method:

1. Select monoclonal strains to LB plate with chl resistance, and carry out PCR after culture for 2h
2. PCR

PCR system（10ul）：

5xl Taq Buffer	2 µL
dNTP	0.8ul µL
Taq polymerase	0.2 µL
Forward primer	0.5 µL
Reverse primer	0.5 µL
template	1 µL
ddH2O	5 µL

PCR procedure


3. Examine the results using electrophoresis. If positive, inoculate 5mL to LB plat with chl resistance for overnight culture. Preserve the stains with 15% glycerol.

Materials:

Rhodobacter sphaeroides 2.4.1
Ecoli-sm10
Joint film
LB medium
PBS buffer

Method:

1. Ecoli-sm10, Rhodobacter sphaeroides 2.4.1 were incubated overnight for 12 h. Add the corresponding antibiotics to donor bacteria .
2. Ecoli-sm10, Rhodobacter sphaeroides 2.4.1 secondary culture, add the appropriate antibiotic into the donor bacteria medium, shake to the logarithmic growth phase.
3. Take Ecoli-sm10, Rhodobacter sphaeroides 2.4.1, each 1ml wash with PBS and mix them, take 25μl drop in the dried adhesive film, dry. Cultivate overnight.
4. Wash the bacteria moss on the joint membrane with 1 ml of medium or PBS and take 100 μl of the coated plate. The results were observed after several hours of culture.
5. After one screening, it was induced with TSB containing 10% sucrose and then subjected to secondary screening on a plate containing 10% sucrose free of resistance.
6. Validate gene knockout and gene knocking by priming pairs on the genome.

Materials:

5xl Gxl Buffer
Primer star Gxl（DNA polymerase）
dNTP
ddH₂O
pIND4 plasmid
pMCS-eq plasmid

Forward/Reverse Primer：

Method:

Mentioned above.

Mentioned above.

Materials:

Enzyme NcoI and BamHI
10xBuffer（NEB buffer 1.1 2.1 3.1 star）
PCR DNA
ddH₂O
pIND4 plasmid DNA

Method:

1. Configuration of double enzyme digestion system(50ul)：

NcoI 1ul
BamHI 1ul
10xBuffer 5ul (NEB's buffer score 1.1 2.1 3.1 star specific see enzyme instructions select the cutting efficiency and asterisk the best activity)
ddH₂O is added as appropriate
DNA 1ug


2. the reaction temperature of 37 ℃ 15min (specific reaction time see the instructions).
3. After the end of the reaction 80 ℃ 20min inactivation.

Materials:

T4 DNA ligase
10x buffer
Plasmid DNA
PCR DNA
ddH₂O

Method:

1. According to the external source and carrier concentration ratio of 5: 1 configuration connection system 20ul

system 20ul
10xbuffer 2ul
T4 DNA ligase 1ul
plasmid DNA Xul
PCR DNA Yul


2. 16 ℃for 1 h
3. The connection solution to take 10ul to transform, with kanr resistance screening positive bacteria

Materials:

10% glycerol
2mm Electroporation cup
Kanr plate
ddH₂O

Method:

1. 32℃ overnight culture and transfer, until the OD grows to about 2.5
2. 10 ml of bacteria at 5000 rpm for 5 min to collect all the cells
3. 2ml sterile water pumped retrogradesum (washed away from the medium ion residue, centrifuged at 5000 rpm for 2 min)
4. with 200ul 10% glycerol resuspend the bacteria
5. 100ul of competent and 100ng plasmid mixed
6. by adding 2mm electric rotor 1.5Kv shock (voltage index curve mode, capacitance 25uF, resistance 200Ω)
7. 600 ulTSB mixed and moved into the EP tube
8. 32℃, 220rpm recovery 2h
9. 5000 rpm after centrifugation, remove the 400ul supernatant, the remaining bacteria coated on kanr resistant plate
10. culture 2-3d observation results

Materials:

Media for fermentation ：
For 1L media:
Succinic acid disodium salt 5.49g
NaCl 0.4g
MgS04·7H20 0.2g
CaCL2·2H20 0.05g
L-glutamate 5mol
KH2PO4 1.0g
K2HPO4 ·2H20 1.572g
Yeast extract fermentation 1.0g
Solution of trace element 1.0ml
Solution of growth factor 1.0ml
For 100ml solution of trace element:
0.21 g MnSO4 · 4H2O
0.28 g H3BO3
0.004 g Cu(NO3)2 · 7H2O,
0.024 g ZnSO4 · 7H2O,
0.075 g Na2MoO4 · 2H2O


For 100ml solution of growth factor:
0.01g biotin
0.5g thiamine HCl
1.0g nicotinic acid

Main protocol:

1. Rhodobacter sphaeroides 2.4.1 were cultured in TSB medium for 48h
2. Took 10ml bacterial suspension and removed the clear supernatant extract after demission at 3500rpm for 5 minutes
3. Resuspended the bacteria in 10ml fermentation medium and transferred them into a new conical flask
4. Added 90ml fermentation medium into the flask with 100μL solution of growth factor, 100μL solution of trace element, 30ng/μL kanamycin and 100μL IPTG (making the final concentration 800μL)
5. Introduced argon to the flask through rubber tube for 5 min and wrapped the bottleneck with plastic membrane in case of gaseous escape
6. Pressed the bottleneck with rubber stopper and sealed it with parafilm
7. Cultured the bacteria in the flask which was exposed to 4,000lx under anaerobic condition with nutrient at 32℃
8. Collected the gas based on water gas displacing principle using pinhead through rubber stopper and the pinhead should be covered with Vaseline
9. Drew bacterial suspension with syringe and measured OD every 24 hours. After each measurement, the pinhead should be covered with Vaseline.