Construction of mutant library
Shuning Guo
Abstract
This protocol is used to construct mutant library of target gene with high efficiency and low false positives/negatives rate after subsequent functional screening.
Before start
Make sure that the template of MEGAWHOP PCR is fresh to improve the construction efficiency.
Steps
Error-prone PCR
Add the following reagent to a PCR tube (50μl) (Random Mutagenesis Kit by Solarbio).
| A | B |
|---|---|
| Template(10μl) | Depends on the concentration |
| Forward Primer (10 μM) | 1μl |
| Reverse Primer (10 μM) | 1μl |
| Mut Enhencer | 3μl |
| 2 x Mut Random System | 25μl |
| ddH2O | Add to 50μl |
Program the thermocycler as follows:
| A | B |
|---|---|
| Temperature | Time |
| 95℃ | 2min |
| 94℃ | 30 s |
| Tm-3~5℃ | 1min |
| 72℃ | 1kb/min |
| 72℃ | 7 min |
| 16℃ | ∞ |
Use the palm centrifuge to mix the solution in PCR tube.
Put the PCR tube into the thermocycler and Run the program.
Using agarose gel electrophoresis to confirm if correct construct was present.
PCR product purification
PCR product purified by E.Z.N.A.® Cycle Pure Kit.
Test the concentration and purity of DNA using NanoDrop.
MEGAWHOP PCR
Add the following reagent to a PCR tube (50μl).
| A | B |
|---|---|
| Plasmid templete (50μl) | Depends on the concentration |
| Purified Production of error-prone PCR (mega primer) (500μl) | Depends on the concentration |
| 2×High Fidelity Master Mix (Enzyme) | 25 μl |
| ddH2O | Add to 50μl |
Program the thermocycler as follows:
| A | B |
|---|---|
| Temperature | Time |
| 95℃ | 5min |
| 95℃ | 30s |
| Depends on the Tm | 30s |
| 72℃ | 2kb/min |
| 72℃ | 7 min |
| 16℃ | ∞ |
Use the palm centrifuge to mix the solution in PCR tube.
Put the PCR tube into the thermocycler and Run the program.
Using agarose gel electrophoresis to confirm if correct construct was present.
PCR product purified by E.Z.N.A.® Cycle Pure Kit.
Test the concentration and purity of DNA using NanoDrop.
DpnI digestion
Add the following reagents to a PCR tube (e.g. 20μl).
| A | B |
|---|---|
| DpnI (NEB) (20,000units/ml) | Depends on the quality of DNA (20units DpnI degests 1μg DNA) |
| 10xCutsmart | 2μl |
| Purified Production of MEGAWHOP PCR | Moderate (e.g.400 ng) |
| ddH2O | Add to 20μl |
Use the palm centrifuge to mix the solution in PCR tube.
Incubate at 37°C for 2 hours and heat Inactivation 80°C for 20 min.
Digestion product purification
Digestion product purified by E.Z.N.A.® Cycle Pure Kit.
Test the concentration and purity of DNA using NanoDrop.
Nick ligation (T4 ligase)
Add the following reagents to a PCR tube (e.g. 20μl)
| A | B |
|---|---|
| T4 DNA ligase (Thermo Fisher) (Weiss U) | 1U |
| Purified Production of DpnI digestion | 50ng |
| 10X T4 DNA Ligase Buffer (Thermo Fisher) | 2μl |
| ddH2O | Add to 20μl |
Use the palm centrifuge to mix the solution in PCR tube.
Incubate the reaction at 16℃ overnight.
Transformation
Transform the nick ligation product into competent cells.
