Cloning of a PCR Fragment into an Expression Vector
This protocol is for cloning PCR product directly into an expression vector such as pET28b.
1. Run PCR amplification using Q5 or Phusion Buffer or Theromfisher SuperFi II. 20 to 50 ul total volume
2. After PCR reaction, add 1 ul of restriction enzymes such as Nde1 and Xho1, incubate at 37 oC for 1 hour to 2 hours.
Check enzyme activity in PCR buffers
| RE | Phusion Buffer | Q5 Buffer |
| NdeI |
+++ |
++ |
| XhoI |
+++ |
<+ |
| BamHI-HF |
- |
<+ |
| BamHI |
+++ |
<+ |
| EcoRI-HF |
+ |
<+ |
| EcoRI |
+++ |
<+ |
| EagI-HF |
+ |
<+ |
| EagI |
+++ |
+++ |
| NheI-HF |
||
| NheI |
<+ |
<+ |
| NotI-HF |
||
| NotI |
+ |
<+ |
| PstI |
+ |
+ |
| SalI-HF |
||
| SalI |
++ |
+ |
| XbaI |
<+ |
- |
| BglII |
++ |
+ |
| EcoRV-HF |
<+ |
<+ |
| EcoRV |
+ |
<+ |
| HindIII-HF |
||
| HindIII |
+ |
<+ |
| KpnI |
+ |
++ |
| NcoI-HF |
||
| NcoI |
+ |
<+ |
If restriction enzyme are not compatible with PCR buffer, gel purify the PCR product and then do digestion.---> perform digestion at step 13
Also digest vector with the same restriction enzymes
3. Add 6X GRGreen loading dye, load to agarose gel.
4. Check PCR product purity. Cut out desired PCR band from gel, add 3 vol of QG buffer.
5. Also cut out vector band, add 3X volumne (of the gel) of QG Buffer
6. Dissolve the gel using hot water (heat a small tray of hot tap water by microwave, don't put these tubes in microwave)
7. Mix vector and insert (PCR product) solution before loading to silica spin column
8. After solution is cooled to ~ room temperature, transfer solution to a silica column.
9. Spin, remove flow through
10. Wash column with 2 X 350 ul PE.
11. Spin additional minute at max speed
12. Elute DNA using 20 ul ddH2O
13. If restriction digestion is needed, add digestion buffer and enzymes and digest at appropriate temperature, such as 37 oC for 1 hour. heat inactivate restriction enzymes. Cool on ice
14. Add 3 ul 10 X T4 ligase buffer, mix
15. Add 1 ul T4 lDNA igase
16. Ligation at room temperature for 30 min to 1 hour.
17. Transform NEBTurbo or other competent cells
18. Plate on LB/antibiotics plate.
19, Grow in a incubator at 37 oC o/n
20. Pick colonies and do PCR screening
21. Grow positive clones in 5 ml LB/antibiotics medium
22. Make miniprep
23. Restriction digestion
24. Sequencing
25. Make glycerol stock.
26. Print labels, store DNA at -20 oC, store glycerol stock at -80 oC.