Team:UESTC-China/Protocol

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Revision as of 05:15, 25 September 2014

UESTC-China

DNA ligation with T4 DNA Ligase
Component 20ul Reaction
1.33X Master Mix 15ul
Insert DNA the moles of insert DNA to vector DNA is 5:1
>Vector DNA 50ng
ddH2O To 20ul
Incubate at 50℃ for 1 hour
DNA ligation with Gibson Assemble
Component 20ul Reaction
Vector DNA 50ng
Insert DNA the moles of insert DNA to vector DNA is 5:1
10X T4 DNA ligase buffer 2ul
T4 DNA ligase 1ul
ddH2O to 20ul
Incubate at 25℃ for 12 hours
Double Digestion of DNA
Component 50ul Reaction
10x FastDigest buffer 5ul
Restriction enzyme 1 1ul
Restriction enzyme 2 1ul
DNA 1-2ug
ddH2O to 50ul
Incubate at 37℃ for 2 hours
PCR protocol for KOD-Plus-Neo
Component 50ul Reaction
ddH2O 33ul
10X buffer for KOD-Plus-Neo 5ul
MgSO4 1ul
10mM dNTPs 1ug
10uM forward primer 1ul
10uM reverse primer 1ul
Template 1ul
KOD enzyme 1ul
Temperature Time Cycle
Step1 94℃ 30s X35 cycles
56℃ 30s
68℃ 30s/kb
Step3 68℃ 5min X1 cycles
10℃ 10min
10uM reverse primer 1ul ddH2O 33ul
PCR protocol for KOD-Plus-Neo
Component 50ul Reaction
ddH2O 32ul
10X buffer for KOD-Plus-Neo 5ul
MgSO4 3ul
10mM dNTPs 5ul
10uM forward primer 1ul
10uM reverse primer 1ul
Template 1ul
KOD enzyme 1ul
Each template will be diluted by the number of moles into 2-10ng/ul.
Temperature Time Cycle
Step1 94℃ 5min X1 cycle
Step2 94℃ 30s X35 cycles
56℃ 30s
68℃ 30s/kb
Step3 68℃ 5min X1 cycles
10℃ 10min
Colony PCR with Taq DNA polymerase
Component 25ul Reaction
ddH2O 15.8ul
10X Taq buffer 2.5ul
2.mM dNTPs 0.5ul
10uM forward primer 0.5ul
10uM reverse primer 0.5ul
Taq enzyme 0.2ul
Water with colony 5ul
Temperature Time Cycle
Step1 95℃ 5min X1 cycles
Step2 94℃ 30s X35 cycles
56℃ 30s
72℃ 1min/kb
Step3 72℃ 10min X1 cycles
10℃ 10min

E. coli Calcium Chloride competent cell protocol

1)Streak E.coli cells (DH5a) on an LB plate; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol)
2) Allow cells to grow at 37℃ overnight
3)Place one colony in 10 ml LB media (+antibiotic selection if necessary), grow overnight at 37℃
4) Take 2 ml LB media and save for blank. Transfer 5 ml overnight DH5a culture into 500 ml LB media in 3 L flask
5) Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (~2-3 hours)
6) Transfer cells to 2 centrifuge bottles (250 ml), and place cells on ice for 20 min
7) Centrifuge cells in at 4oC for 10 min at 3,000 g and subsequent resuspension may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room
8) Pour off media and resuspend cells in 30 ml of cold 0.1 M CaCl2. Transfer the suspended cells into 50 ml polypropylene tubes, and incubate on ice for 30 min
9) Centrifuge cells at 4O℃ for 10 min at 3,000 g
10) Pour supernatant and resuspend cells (by pipetting) in 8 ml cold 0.1M CaCl2 containing 15% glycerol. Transfer 140 ml into (1.5 ml) Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80oC can be used for transformation for up to ~6 months
11) Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step
12) Incubate the mixture on ice for 30 minutes.
13) Transfer the reaction to a 42℃ water for 1min.
14) Add 0.9 ml of LB culture to each tube and incubate at 37℃ for 1 hour in a roller drum (250 rpm) to allow cells to recover and express the antibiotic resistance marker.
15) Incubate on ice for 2 minutes.
16) Spread the appropriate quantity of cells (50 to 100 ml) on selective media. Store the remaining cells at 4℃.
(A) E. coli cells from the control tube without DNA in step 12 above are plated on selective medium and nonselective medium. The first plating ensures that the selective medium is working properly since no growth should be observed. The second plating provides the number of viable cells in the absence of selective medium.
(B) E. coli cells being tested for competency are plated on LB agar containing ampicillin (50 mg/ml final concentration) to ensure that the transformation efficiency has not decreased over time due to storage.
17) Incubate all plates overnight at 37℃ (agar side up).
18) Count the number of colonies.

Agrobacterium tumefacien EHA105 Mediated Transformation with freeze-thawing steps

(1)Have 0.5~1 g plasmid DNA into 100 L competent Cells,on ice for 30 min;
(2)Frozen in liquid nitrogen for 5 min, grow at 37 C for 5 min,on ice for 2 min;
(3)Pour in 1000 LYEB,200 rpm、grow at 28 C for 2~3 hr;
(4)6000rpm,2min. Suspend collected bacteria with 100ul YEB and evenly coat that at a YEB medium. (125 mg/L Sm or 50 mg/L rif, and 50 mg/L Kan included);
(5)Grow upside down at 28℃,for 48h。
(6)Pick positive clones and grow them in LB medium with antibiotic at 28℃for 48h。
(7)Inject to LB medium in flasks by the day of transformation, in the rate of 1:50. Grow to OD600 = 0.5。Ready to infect tobacco leaf discs.

Agrobacterium-mediated genetic transformation to tobacco

Tobacco was transformed essentially by following the leaf disk co-cultivation protocol of Horsch et al[1]. Co-cultivation was initiated by dipping leaf disks in an Agrobacterium suspension, blotting them on sterile tissue paper, and incubating them for 2 d on MS medium (Murashige and Skoog 1962 ) containing naphthalene acetic acid (NAA 0.1mg/L), 6-Benzylaminopurine (6-BA,2.0mg/L). Cefotaxime sodium (Cef) was included in the medium (500mg/L) to inhibit Agrobacterium growth. The leaf disks were then transferred onto a medium containing antibiotics for transgenic plant selection(kanamycin, 50 mg/L), and NAA (0.1 mg/L), 6-BA (2.0 mg/L), Cef (500mg/L). And incubate them for 1 month on the medium above. At last, cut off the bud from the callus, put the buds into the mudium containing NAA (0.1mg/L), Cef (500mg/L) and kanamycin (25 mg/L).

detection method

1)Molecular identification
Extract Genomic DNA from leaves of tobacco seedlings. Using specific primers to amplify the target gene. Extract leaf mRNA from leaves of grown tobacco which DNA testing was positive. Detected by the method of RT-PCR whether target gene is expressed.
2)Phenotype testing

The experimental group control group
Qualitative detection Have 6 positive seedlings from every strain(about 8 leaves age) , and 6 wild-type seedings with the same growth equally distributed into 3 650ml culture bottles. Treat with 10ul 37% formaldehyde for a week.
Quantitative detection Have 4 positive seedlings from every strain(about 8 leaves age) in a 650ml culture bottle. Treat with 10ul 37% formaldehyde for 2 weeks. Using a HCHO detector (Gastec Passive Dositube, 91D, Ayase, Kanagawa, Japan) was set in the hole to detect gaseous HCHO. Three and a half hours later, the measurement was stopped and the results were photographed[2]. Put 4 wild -type seedlings with the same growth of seedlings in experimental group into 650ml culture bottle , with same processing as the case of the experimental group .

References
[1] Horsch RB, Fry JE, Hoffmann NL, Eichholts D, Rogers SG, and Fraley RT, Science,227, 1229-1231 (1985).
[2] Li-mei CHEN et al, Biosci. Biotechnol. Biochem, 74(3), 627-635 (2010)