Phusion Site-Directed Mutagenesis to make plasmids Golden Gate Cloning Ready

General layout of desired product

Sense strand: 30-35 bp homology – introduced sequence – 8bp spacer – introduced sequence – 30-35 bp homology

To make a plasmid compatible for Golden Gate Cloning, this takes the form of:
Sense strand: 5’ upstream region of homology – upstream new sequence (optional) – GG sticky end start – spacer – comp. rev. BsaI – linker– BsaI – spacer – GG sticky end end- downstream new sequence (optional)- downstream region of homology- 3’

Recommended Constants for Golden Gate Cloning:
1) Keep the linker constant.
2) The location and orientation of the BsaI sites must be kept constant for Golden Gate Cloning to work.

Choices to be made for Golden Gate cloning:

1. At what upstream and downstream bp do you want to insert your new sequence? Are there any intervening regions that you want to delete? This dictates where the primers bind to the template (green regions).
2. Is there any sequence you want to insert between the homology region and the GG sticky ends?

Procedure for Design

1. Use VectorNTI of original plasmid to decide where to insert new sequence and any sequence to delete. Notate these regions (e.g., regions to be deleted for pAF000X, insert new sequence here for pAF000X) in your VectorNTI file
2. Draw block diagram of new sequence to lie between these regions in powerpoint or word.
3. Modify the sequence of your original plasmid so that it incorporates these new regions. These are the regions you will insert into your plasmid using the Phusion-based mutagenesis, so notate them accordingly (‘regions to be inserted’). Save this new Vector NTI file with a pAF000X name.
4. List start of this region and end of this region:____ & _____. Subtract 40bp from the start site and add 40bp to the end site.
5. Copy the both the sense and antisense strands from new region + 40 bp upstream and 40 bp downstream from your pAF000X file into in word or ppt. Notate these using your color coding
6. Use the ppt/doc file to identify the regions that you will attach to the 5’ end of your primers.
7. Use Vector NTI to design primers that have Tm ~ 60-65C. Highlight everything except the regions to be deleted and label this as “region to be amplified’.
8. If the 5′ flanking regions (5′ regions to attach to primer) are long, the homologous region may need to be long as well.

Procedure

1. Set up Phusion-based pcr. Mix:
   • 1 uL 10 uM forward primer
   • 1 uL 10 uM reverse primer
   • 1 uL plasmid template (1pg-10 ng)
   • 10 uL 5x HF buffer (unless GC content is high, then 5xGC buffer)
   • 1 uL 10 mM dNTPs
   • 1 uL DMSO
   • water to 49.5 uL
   • 0.5 uL Phusion enzyme
2. Run the PCR. The cycle should be:
   

   Step	Temperature (C)	Time(s)
   Initial Denature	98	30
   Denature	98	8
   Anneal	VectorNTI or NEB site	30
   Extend	72	15-30s/1kB
   Repeat denature-extend	35 times
   Final Extend	72	8 min

   Note: I’ve found running a couple reactions with different annealing temperatures helps identify at least 1 condition that gives good amplification. Neither the VectorNTI or NEB site recommended temperatures have been consistently spot on for me.

3. PCR purify the reactions
4. Check for the correct length via DNA gel electrophoresis
5. Digest with DpnI to remove the template DNA. Mix:
   • 25 uL PCR product
   • 3 uL 10xNEB 4
   • 1 uL water
   • 1 uL DpnI at 2U/uL

   Incubate at 37C for 1 hr. Inactivate DpnI by heating to 80C for 20 min.

6. Do a blunt-end ligation. Mix:
   • 25 ng PCR product+ water, to 5 uL
   • 5 uL 2x Quick Ligase Buffer
   • 0.5 uL Quick T4 DNA Ligase

   Incubate at room temperature for at least 15 min.

7. Transform into Mach 1 chemically competent cells. Typically 2 uL ligation mix + 20 uL competent cells gives tens-hundreds of transformants.
8. Procedure to plasmid verification.