Team:ZJUT-China/Protocols2018
Team:ZJUT-China
PCR using Phanta Max Master Mix
Methods:
1. Prepare the following reaction system on ice;
| ddH2O | to 50µl |
|---|---|
| 2×Phanta Max Master Mix | 25µl |
| DNA template（20~40ng） | 1µl |
| Primer Forward(10µM) | 2µl |
| Primer Reverse(10µM) | 2µl |
2.Mix gently, collect liquid at the bottom by centrifuging for a few seconds;
3.Start Polymerase Chain Reaction
| PCR condition setting | ||
|---|---|---|
| Step one | 95℃ 3min | |
| Step two | 95℃ 15sec | 30-35 cycles |
| Step three | T*℃ 15sec | 30-35 cycles |
| Step four | 72℃ 60sec/kb | 30-35 cycles |
| Step five | 72℃ 5min |
DNA purification
(Refer to the AxyPrep PCR Clean-Up Kit)
Methods:
1. Add a 3× reaction volume of Buffer PCR-A to the sample. If the required volume of Buffer PCR-A is less than 100 μl, add 100 μl of Buffer PCR-A. Vortex briefly to mix the contents.
2. Place a PCR column into a 2 ml Microfuge tube. Pipette the reaction from Step1 into the PCR column. Centrifuge at 12000×g for 1 minute.
3. Discard the filtrate from the 2 ml Microfuge tube. Return the PCR column to the 2 ml Microfuge tube. Pipette 700 μl of Buffer W2 into the column and centrifuge at 12000×g for 1 minute. Note: Make sure that the volume of ethanol specified on the bottle label has been added to the Buffer W2 concentrate.
4. Discard the filtrate. Return the PCR column to the 2 ml Microfuge tube. Pipette 400 μl of Buffer W2 into the column and centrifuge at 12000×g for 1 minute.
5. Transfer the PCR column into a clean 1.5 ml Microfuge tube (provided). To elute the DNA, add 25-30 μl of Eluent (pre-warmed at 65°C) to the center of the membrane. Let it stand for 1 minute at room temperature. Centrifuge at 12000×g for 1 minute.
Colony PCR
Methods:
1.Pick a single colony and add it to the PCR tubule containing 20µl ddH2O.
2.Boil bacteria solution at 100 ℃ water bath about 10-15min.
3.Prepare the PCR mixture (50µl system)
| ddH2O | to 50µl |
|---|---|
| Green Taq Mix | 20µl |
| Boiled bacteria solution | 1µl |
| Primer Forward(10µM) | 2µl |
| Primer Reverse(10µM) | 2µl |
4.Start Polymerase Chain Reaction
| PCR condition setting | ||
|---|---|---|
| Step one | 94℃ 5min | |
| Step two | 94℃ 30sec | 30-35 cycles |
| Step three | 58℃ 30sec | 30-35 cycles |
| Step four | 72℃ 60sec/kb | 30-35 cycles |
| Step five | 72℃ 7min |
5.Valid PCR products by gel electrophoresis
Preparation of Escherichia coli competent cells
(CaCl2 method)
Methods:
1.Select a single colony from the newly activated bacteria plate and vaccinate it in LB liquid culture of 3~5ml for about 12 hours at 37 ℃ until it get logarithmic growth phase.
2.Transfer the suspension to the 100ml LB liquid medium at 1: 100 to 1: 50 and oscillate at 37 ℃ to amplification. When the culture medium begin to be muddy, measure the OD600nm every time at intervals of 20~30min, and stop the culture at OD600nm ≤ 0.5.
3.Transfer three 2ml in each group into the 2ml centrifuge tube, cool them on ice for 20-30min, then centrifuge them for 10min at 4 ℃, 4000rp / min.
PS:·From this step on, all operations should be performed on the ice, as fast and steady as possible.
4.Empty supernatant medium, gently suspend cells with 1ml cold CaCl2 solution(0.1mo1/L), then ice bath.
5.4℃, 4000r/min, centrifuge for 10min.
6.Empty supernatant, add 500µl cold CaCl2 solution, suspend cells carefully. Then, 4℃, 4000r/min, cenreifuge for 10min.
7.Empty supernatant, add 500µl cold CaCl2 solution, suspend cells carefully. After being placed on the ice for a short time, the competent cells are made.
8.The competent cells can be used in transformation experiment directly, or it can be added to 30% glycerol sterilized by high pressure, mixed evenly in the 1.5ml centrifuge tube, placed at -70℃, and can be preserved for half a year to one year.
Chemical Transformation
Methods:
1.Thaw 50µl DH5α competent E. coli cells and a ligation reaction mix on ice.
2.Add 5µl DNA from a ligation reaction mix to 50µl DH5α competent E. coli cells
3.Place the mixture on ice for 30min.
4.Heat shock at 42°C for 90sec.
5.Place on ice for 1-2min.
6.Pipette 945μl of 37℃ pre-warmed LB medium into the mixture.
7.Incubate at 37°C for 60min.
8.Centrifuge at 6000 rmp for 5min, remove 700μl of culture solution, then mix the residue.
9.Add 30µl of the transformed cells to the selection plate.
LB Broth
Methods:
1.Mix the components listed.
| Component | 1000ml |
|---|---|
| Tryptone | 10g |
| Yeast Extract | 5g |
| NaCl | 10g |
| Sterile water | to 1000ml |
2.Autoclave.
LB Ager
Methods:
1.Mix the components listed.
| Component | 1000ml |
|---|---|
| Ager | 20g |
| Tryptone | 10g |
| Yeast Extract | 5g |
| NaCl | 10g |
| Sterile water | to 1000ml |
2.Autoclave.
Basic salt medium
Methods:
1.Mix the components listed.
| Component | 1000ml |
|---|---|
| K2HPO4·3H2O | 14g |
| KH2PO4 | 5.2g |
| (NH4)2SO4 | 2g |
| MgSO4 | 0.3g |
| Tryptone | 1g |
2.Autoclave.
3.Add 10g of glucose to the medium when using.
Plasmid Extraction
(Refer to the AxyPrep Plasmid DNA Mini Kit)
Methods:
1.Prepare 1-4mL bacteria solution grown overnight in LB broth, centrifuge at 12,000×g for 1min. Discard the supernatant.
2.Add 250µl Buffer S1 to suspend bacteria cells and transfer to a 1.5mL centrifuge tube. Please make sure that Buffer S1 has been added RNase A.
3.Add 250µl Buffer S2 and gently invert the tube 4-6 times to mix. This step should not exceed 5 minutes. And the bottle with Buffer S2 should be closed immediately after using.
4.Add 350µl Buffer S3 and gently invert the tube 6-8 times to mix.
5.Centrifuge for 10min at 12,000×g.
6.Apply the supernatants from step 5 to the spin column by pipetting. Centrifuge for 1min. Discard the filtrate.
7.Wash the spin column by adding 500µl Buffer W1, and centrifuging for 1min. Discard the filtrate.
8.Wash the spin column by adding 700µl Buffer W2, and centrifuging for 1min. Discard the filtrate. Please make sure that Buffer W2 has been added anhydrous ethanol of specified volume
9.Repeat the step 8.
10.Centrifuge for an additional 2min to remove residual wash buffer.
11.Place the spin column in a clean 1.5ml centrifuge tube. Add 60-80µl ddH2O (65℃) to the center of the spin column, let stand for 1min, and centrifuge for 1min at 12,000×g.
One Step Cloning
(Refer to the Hieff CloneTM Multi One Step Cloning Kit)
Methods:
1.Prepare the linearized cloning vector.
2.Prepare PCR products.
2.1 Design primers.
2.2 PCR
3.Recombine.
3.1 Prepare the reaction system.
| ddH2O | up to 20 μl |
|---|---|
| 5×CE MultiS Buffer | 4μl |
| linearized cloning vector | X ng |
| PCR products | X ng |
| Exnase MultiS | 2μl |
PS: ·The reaction system was prepared above ice water bath.
·Optimal use per segment = (0.02×base pairs)ng
·The usage of the linearized cloning vector should be between 50~200ng and that of insert DNA should be over 10ng.
3.2 Begin recombination reaction: when finishing preparing the system, gently blow to mix the ingredients and avoid bubbles. After reacting for 20~50min at 50℃, place the reaction tube over ice water bath immediately for 5min to get final products.
