Protocol
Making and Running Agarose Gels
- 1.Add the TAE buffer.
- 2. Add the agarose powder with a volume to the mass ratio of X % and add it into the buffer.
- 3. The mixture is heated several times in a microwave oven until the solution becomes clear.
- 4. Cool the solution to about 40-50°C and add the nucleic acid dye.
- 5. Place the appropriate comb on the gel tray and pour the solution into the tray gel and allow the gel to cool until it becomes hard.
- 6. Carefully pull out the comb, do not damage the glue hole, take the gel into the electrophoresis room.
- 8. Add enough TAE buffer to make it no gel.
- 9. Take the appropriate amount of the loading buffer and the DNA (Gene Finder) mixed DNA sample into the pores of the gel; take the appropriate amount of marker into the gel pore as a control.
- 10. Run the gel at 100V, for 60 minutes.
Table.1. correct percent agarose gel concentration for resolving DNA fragments.
Percent Agarose gel (w/v) | DNA size resolution (bp) |
---|---|
0.5 % | 2000-50000 |
0.6 % | 1000-20000 |
0.7 % | 800-12000 |
0.8 % | 800-10000 |
0.9 % | 600-10000 |
1.0 % | 400-8000 |
1.5 % | 200-3000 |
2.0 % | 100-2000 |
3.0 % | 25-1000 |
4.0 % | 10-500 |
5.0 % | 10-300 |
Making Agar Plates
- 1. Prepare LB-Agar for autoclave: 10 g tryptone, 5 g yeast extract and 5 g of NaCl per litre.
- 2. Autoclave. If it is still molten when collected, it can be left in a 60°C water bath to cool down for poouring. Otherwise, if it has solidified, it needs to be microwaved until molten, then left in a 60°C water bath to cool to the correct temperature.
- 3. Once agar is molten, mix carefully. Add antibiotics and any other desired additives.
- 4. Pour plates carefully.
- 5. Allow to set for approximately 10 min.
- 6. Invert plates and allow to dry for 20-30 min.
- 7. Store plates at 4°C
eGFP
- 1. Prepare master mix
Components (50μl)
Volume(μl)
10X Tag Buffer
5
dNTPs (10 mM)
2.5
Template
1
Primer-F
2.5
Primer-R
2.5
Tag DNA Polymerase
1
ddH20
35.5
- 2.Transfer tube to a thermocycler with the following program.
Step
Temperature(℃)
Time(s)
initial denaturing
95
300
10 cycles
95
30
10 cycles
52
30
10 cycles
72
30
30 cycles
72
30
30 cycles
60.5
30
30 cycles
72
30
extension
72
420
Sec VMA intein
- 1. Prepare master mix
Components (50μl)
Volume(μl)
10X Tag Buffer
5
dNTPs (10 mM)
2.5
Template
1
Primer-F
2.5
Primer-R
2.5
Tag DNA Polymerase
1
ddH20
35.5
- 2.Transfer tube to a thermocycler with the following program.
Step
Temperature(℃)
Time(s)
initial denaturing
95
300
10 cycles
95
30
10 cycles
52
30
10 cycles
72
30
30 cycles
72
30
30 cycles
58.5
30
30 cycles
72
30
extension
72
420
Hold
4
forever
SUMO
- 1. Prepare master mix
Components (50μl)
Volume(μl)
10X Tag Buffer
5
dNTPs (10 mM)
2.5
Template
1
Primer-F
2.5
Primer-R
2.5
Tag DNA Polymerase
1
ddH20
35.5
- 2.Transfer tube to a thermocycler with the following program.
Step
Temperature(℃)
Time(s)
initial denaturing
95
300
10 cycles
95
30
10 cycles
52
30
10 cycles
72
30
30 cycles
72
30
30 cycles
60.8
30
30 cycles
72
30
extension
72
420
Hold
4
forever
PCR Amplification
Sequence of primer
Primer
Length
Forward
AAATGGCTGCGTTGGGTCCGCCGCC
25mer
Reverse
CCACCAACGGCGGCGGACCCAACGCAG
27mer
- 1. Prepare master mix
Components (50μl)
Volume(μl)
10X Tag Buffer
5
dNTPs (10 mM)
5
Primer-F
5
Primer-R
5
Tag DNA Polymerase
1
ddH20
29
- 2.Transfer tube to a thermocycler with the following program.
Step
Temperature(℃)
Time(s)
initial denaturing
94
300
30 cycles
94
30
30 cycles
59
30
30 cycles
70
30
extension
72
300
Hold
4
forever
Overlap Extension PCR:
(TAPG)
- 1. Prepare master mix
Components (50μl)
Volume(μl)
10X Tag Buffer
5
dNTPs (10 mM)
2.5
DNA Template
1
Primer-F
2.5
Primer-R
2.5
Tag Polymerase
1
ddH20
35.5
- 2.Transfer tube to a thermocycler with the following program.
Step
Temperature(℃)
Time(s)
initial denaturing
95
300
10 cycles
95
30
10 cycles
50
30
10 cycles
72
30
30 cycles
72
30
30 cycles
60.8
30
30 cycles
72
30
extension
72
420
Hold
4
forever
Overlap Extension PCR:
- 1. Prepare master mix
Components (50μl)
Volume(μl)
10X Tag Buffer
5
dNTPs (10 mM)
5
DNA Template
1
Primer-F
2.5
Primer-R
2.5
Tag Polymerase
1
ddH20
33
- 2.Transfer tube to a thermocycler with the following program.
Step
Temperature(℃)
Time(s)
initial denaturing
94
300
10 cycles
94
30
10 cycles
50
30
10 cycles
72
30
30 cycles
94
30
30 cycles
59
30
30 cycles
70
30
extension
72
300
Hold
4
forever
PCR(check)
- 1. Prepare master mix
Components (50μl)
Volume(μl)
10X Tag Buffer
5
dNTPs (10 mM)
1
DNA Template
1
Primer-F
1
Primer-R
1
Tag Polymerase
1
ddH20
40
- 2.Transfer tube to a thermocycler with the following program.
Step
Temperature(℃)
Time(s)
initial denaturing
94
300
30 cycles
94
30
30 cycles
51
30
30 cycles
70
30
extension
72
300
Hold
4
forever
Colony PCR
- 1. Prepare master mix
Components (50μl)
Volume(μl)
10X Tag Buffer
5
Insert Fragment
1
Backbone Fragment
20
T7 DNA ligase
1
Bsa I –HFv2
1
ddH2O
22
Ligation
- 2.Incubate ligations at 4°C overnight
- 3.Transform into chemically competent E. coli by heat shock
- 1. Prepare master mix
- 2.Transfer tube to a thermocycler with the following program.
- 1. Prepare master mix
- 2.Transfer tube to a thermocycler with the following program.
- 1. Prepare master mix
- 2.Transfer tube to a thermocycler with the following program.
- 1. Prepare master mix
- 2.Transfer tube to a thermocycler with the following program.
- 1. Prepare master mix
- 2.Transfer tube to a thermocycler with the following program.
- 1. Prepare master mix
- 2.Transfer tube to a thermocycler with the following program.
- 1. Prepare master mix
- 2.Transfer tube to a thermocycler with the following program.
- 1. Prepare master mix
- 2.Incubate ligations at 4°C overnight
- 3.Transform into chemically competent E. coli by heat shock
Components (50μl) | Volume(μl) |
---|---|
10X Tag Buffer | 5 |
dNTPs (10 mM) | 2.5 |
Template | 1 |
Primer-F | 2.5 |
Primer-R | 2.5 |
Tag DNA Polymerase | 1 |
ddH20 | 35.5 |
Step | Temperature(℃) | Time(s) |
---|---|---|
initial denaturing | 95 | 300 |
10 cycles | 95 | 30 |
10 cycles | 52 | 30 |
10 cycles | 72 | 30 |
30 cycles | 72 | 30 |
30 cycles | 60.5 | 30 |
30 cycles | 72 | 30 |
extension | 72 | 420 |
Sec VMA intein
Components (50μl) | Volume(μl) |
---|---|
10X Tag Buffer | 5 |
dNTPs (10 mM) | 2.5 |
Template | 1 |
Primer-F | 2.5 |
Primer-R | 2.5 |
Tag DNA Polymerase | 1 |
ddH20 | 35.5 |
Step | Temperature(℃) | Time(s) |
---|---|---|
initial denaturing | 95 | 300 |
10 cycles | 95 | 30 |
10 cycles | 52 | 30 |
10 cycles | 72 | 30 |
30 cycles | 72 | 30 |
30 cycles | 58.5 | 30 |
30 cycles | 72 | 30 |
extension | 72 | 420 |
Hold | 4 | forever |
SUMO
Components (50μl) | Volume(μl) |
---|---|
10X Tag Buffer | 5 |
dNTPs (10 mM) | 2.5 |
Template | 1 |
Primer-F | 2.5 |
Primer-R | 2.5 |
Tag DNA Polymerase | 1 |
ddH20 | 35.5 |
Step | Temperature(℃) | Time(s) |
---|---|---|
initial denaturing | 95 | 300 |
10 cycles | 95 | 30 |
10 cycles | 52 | 30 |
10 cycles | 72 | 30 |
30 cycles | 72 | 30 |
30 cycles | 60.8 | 30 |
30 cycles | 72 | 30 |
extension | 72 | 420 |
Hold | 4 | forever |
PCR Amplification
Sequence of primer
Primer | Length | |
---|---|---|
Forward | AAATGGCTGCGTTGGGTCCGCCGCC | 25mer |
Reverse | CCACCAACGGCGGCGGACCCAACGCAG | 27mer |
Components (50μl) | Volume(μl) |
---|---|
10X Tag Buffer | 5 |
dNTPs (10 mM) | 5 |
Primer-F | 5 |
Primer-R | 5 |
Tag DNA Polymerase | 1 |
ddH20 | 29 |
Step | Temperature(℃) | Time(s) |
---|---|---|
initial denaturing | 94 | 300 |
30 cycles | 94 | 30 |
30 cycles | 59 | 30 |
30 cycles | 70 | 30 |
extension | 72 | 300 |
Hold | 4 | forever |
Overlap Extension PCR:
(TAPG)Components (50μl) | Volume(μl) |
---|---|
10X Tag Buffer | 5 |
dNTPs (10 mM) | 2.5 |
DNA Template | 1 |
Primer-F | 2.5 |
Primer-R | 2.5 |
Tag Polymerase | 1 |
ddH20 | 35.5 |
Step | Temperature(℃) | Time(s) |
---|---|---|
initial denaturing | 95 | 300 |
10 cycles | 95 | 30 |
10 cycles | 50 | 30 |
10 cycles | 72 | 30 |
30 cycles | 72 | 30 |
30 cycles | 60.8 | 30 |
30 cycles | 72 | 30 |
extension | 72 | 420 |
Hold | 4 | forever |
Overlap Extension PCR:
Components (50μl) | Volume(μl) |
---|---|
10X Tag Buffer | 5 |
dNTPs (10 mM) | 5 |
DNA Template | 1 |
Primer-F | 2.5 |
Primer-R | 2.5 |
Tag Polymerase | 1 |
ddH20 | 33 |
Step | Temperature(℃) | Time(s) |
---|---|---|
initial denaturing | 94 | 300 |
10 cycles | 94 | 30 |
10 cycles | 50 | 30 |
10 cycles | 72 | 30 |
30 cycles | 94 | 30 |
30 cycles | 59 | 30 |
30 cycles | 70 | 30 |
extension | 72 | 300 |
Hold | 4 | forever |
PCR(check)
Components (50μl) | Volume(μl) |
---|---|
10X Tag Buffer | 5 |
dNTPs (10 mM) | 1 |
DNA Template | 1 |
Primer-F | 1 |
Primer-R | 1 |
Tag Polymerase | 1 |
ddH20 | 40 |
Step | Temperature(℃) | Time(s) |
---|---|---|
initial denaturing | 94 | 300 |
30 cycles | 94 | 30 |
30 cycles | 51 | 30 |
30 cycles | 70 | 30 |
extension | 72 | 300 |
Hold | 4 | forever |
Colony PCR
Components (50μl) | Volume(μl) |
---|---|
10X Tag Buffer | 5 |
Insert Fragment | 1 |
Backbone Fragment | 20 |
T7 DNA ligase | 1 |
Bsa I –HFv2 | 1 |
ddH2O | 22 |
Ligation
Golden Gate Assembly
Components (50μl) | Volume(μl) |
---|---|
Intein | 1 |
SUMO | 1 |
eGFP | 1 |
6x His | 1 |
T7 DNA ligase | 1 |
Bsa I –HFv2 | 1 |
Tag DNA Polymerase | 2.5 |
ddH2O | 16.5 |