<noautolink> ---+ 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 | Part Type | Prefix (F) | Prefix (R) | Suffix (F) | Suffix (R) | | 2 - promoter+RBS | | | | | | 3 - gene | | | | | | 4 - terminator | | | | | *Note: the suffix sequences have been reverse complemented * ---++ 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
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Topic revision: r5 - 2016-05-11 - JeffreyBarrick