Golden Gate: Making A New Part

This protocol describes how to clone a new Golden Gate part amplified from a template DNA or ordered as a gBlock into the YTK001 entry vector using a BsmBI assembly reaction.

Supplies

• Phusion® High-Fidelity DNA Polymerase (NEB: M0530L)
• 10X T4 DNA ligase buffer (NEB: M0202S)
• T7 DNA ligase (NEB: M0318S)
• restriction endonuclease BsaI (NEB: R0535) or BsmBI (NEB: R0580S)
• competent cells
• SOC and liquid media
• LB plates

Step 1: Creating DNA fragment for cloning

Method 1: Ordering a gBlock containing required flanking regions

Method 2: Amplifying a sequence to add required flanking regions

You need to order two primers that each consist of two pieces:

1. The outer piece (A or B) contains a BsmBI cut site (used for cloning at this step) and a BsaI cut site (used in stage 1 Golden Gate assembly)
2. The inner piece (C or D) that that amplifies the part sequence.

Order the (A+C) primer and (B+D) primers designed as shown below.

Primer-F GCATCGTCTCATCGGTCTCA[4-bp Prefix] (A) + 20bp amplifies the Part (C)

Primer-R ATGCCGTCTCAGGTCTCA+4bp TYPE 2/3/4 over overhang (B) + 20bp amplifies the Part(D)

Part Overlap Quick Reference Table

Example

PCR Insert

Use standard 25ul Phusion (or other high fidelity polymerase) protocol

PCR (25ul reaction)

5 ul 5x Buffer

1.5 ul dntps

1.25 ul primer (A+C)

1.25 ul primer (B+D)

x ul template plasmid (<250ng)

ddH20 to 24.5 ul

then add 0.25 ul Phusion

Set elongation time according to size of insert. Purify PCR products

Step 2: Golden Gate Assembly Reaction

Golden Gate Assembly

1. Mix 17.7ng pYTK-001 plasmid and 20 fmol=[(0.02*(660)*(1/(10^6))*insert length)*1000]ng insert of your DNA fragments together.

2. Add that 2μL of 10X T4 DNA ligase buffer, 1 μL of BsmBI, 1 μL of T7 DNA ligase , and add water up to 20 μL, mix well by pipetting.

3. Incubate the reaction at (42°C for 5 min and then 16°C for 5 min) *30 , followed by 10 min at 55°C, 10min at 80 °C .

4. Use 2 µl assembly reaction for electroporation.

Contributors

• Peng Geng
• Jeffrey Barrick