Editing Cloning of a PCR Fragment into an Expression Vector

This protocol is for cloning PCR product directly into an expression vector such as pET28b.&nbsp;

1. Run PCR amplification using '''Q5 or Phusion Buffe'''r 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&nbsp;buffers

[https://www.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-pcr-buffers https://www.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-pcr-buffers]

&nbsp;&nbsp;

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| RE
| style="width: 201px;" | Phusion Buffer
| style="width: 135px;" | Q5 Buffer
|-
| NdeI<br/>
| style="width: 201px;" | +++ &nbsp; &nbsp;<br/>
| style="width: 135px;" | ++<br/>
|-
| <br/>
| style="width: 201px;" | <br/>
| style="width: 135px;" | <br/>
|-
| XhoI<br/>
| style="width: 201px;" | +++ &nbsp; &nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| BamHI-HF<br/>
| style="width: 201px;" | - &nbsp; &nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| BamHI&nbsp;<br/>
| style="width: 201px;" | +++ &nbsp;&nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| EcoRI-HF<br/>
| style="width: 201px;" | + &nbsp; &nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| EcoRI&nbsp;<br/>
| style="width: 201px;" | +++ &nbsp; &nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| EagI-HF<br/>
| style="width: 201px;" | + &nbsp; &nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| EagI<br/>
| style="width: 201px;" | +++ &nbsp; &nbsp;<br/>
| style="width: 135px;" | +++<br/>
|-
| NheI-HF<br/>
| style="width: 201px;" | <br/>
| style="width: 135px;" | <br/>
|-
| NheI<br/>
| style="width: 201px;" | <+ &nbsp; &nbsp; &nbsp; &nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| NotI-HF<br/>
| style="width: 201px;" | <br/>
| style="width: 135px;" | <br/>
|-
| NotI<br/>
| style="width: 201px;" | + &nbsp; &nbsp; &nbsp; &nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| <br/>
| style="width: 201px;" | <br/>
| style="width: 135px;" | <br/>
|-
| PstI<br/>
| style="width: 201px;" | + &nbsp; &nbsp; &nbsp; &nbsp;<br/>
| style="width: 135px;" | +<br/>
|-
| SalI-HF<br/>
| style="width: 201px;" | <br/>
| style="width: 135px;" | <br/>
|-
| SalI&nbsp;<br/>
| style="width: 201px;" | ++ &nbsp; &nbsp; &nbsp;&nbsp;<br/>
| style="width: 135px;" | +<br/>
|-
| XbaI&nbsp;<br/>
| style="width: 201px;" | <+ &nbsp; &nbsp; &nbsp; &nbsp;<br/>
| style="width: 135px;" | -<br/>
|-
| BglII<br/>
| style="width: 201px;" | ++ &nbsp;&nbsp;<br/>
| style="width: 135px;" | +<br/>
|-
| EcoRV-HF<br/>
| style="width: 201px;" | <+ &nbsp; &nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| EcoRV&nbsp;<br/>
| style="width: 201px;" | + &nbsp; &nbsp; &nbsp; &nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| HindIII-HF<br/>
| style="width: 201px;" | <br/>
| style="width: 135px;" | <br/>
|-
| HindIII<br/>
| style="width: 201px;" | + &nbsp;&nbsp;<br/>
| style="width: 135px;" | <+<br/>
|-
| <br/>
| style="width: 201px;" | <br/>
| style="width: 135px;" | <br/>
|-
| KpnI<br/>
| style="width: 201px;" | + &nbsp; &nbsp;<br/>
| style="width: 135px;" | ++<br/>
|-
| NcoI-HF<br/>
| style="width: 201px;" | <br/>
| style="width: 135px;" | <br/>
|-
| NcoI &nbsp;&nbsp;<br/>
| style="width: 201px;" | + &nbsp; &nbsp;<br/>
| style="width: 135px;" | <+<br/>
|}

If restriction enzyme are&nbsp;not compatible with PCR buffer, gel purify the PCR product &nbsp;and then do digestion.---> perform digestion at&nbsp;step 13

Also digest vector with the same restriction enzymes

3.&nbsp;&nbsp;Add &nbsp;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.&nbsp;

5. Also cut out vector band, add &nbsp;3X volumne (of the gel) of QG Buffer

6. Dissolve the gel using hot water (heat a small tray of hot tap&nbsp;water&nbsp;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 &nbsp;solution is cooled to ~&nbsp;room temperature, transfer&nbsp;solution to a silica column.&nbsp;

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&nbsp;ul 10 X &nbsp;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 &nbsp;in a &nbsp;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&nbsp;

25. Make glycerol stock.

26. Print labels, store DNA at -20 oC, store glycerol stock at -80 oC.
[[Category:Protocols]]