Editing GoldenGate TALEN Assembly (section)

=== DAY1<br>  ===

<br>1. Choose your TALEN RVD sequence: N = number of RVDs (12-31) <br><br> 

'''If the TALEN length is 12-21:'''<br>2. Pick plasmids for the RVDs 1-10 (e.g. pNI1, pNN2, pHD3, pHD4....) + destination vector pFUS_A<br>3. Pick plasmids 11 up to N-1 + destination vector pFUS_B#N-1 (pFUS_B plasmids are labeled B1-B10 but they are used for RVDs 11-30 – if<br>the RVD #N-1 is 19 or 29, use the same destination vector pFUS_B9)<br> 

<br>'''If the TALEN length is 22-31:'''<br>2. Pick plasmids for the RVDs 1-10 + destination vector pFUS_A30A, pick plasmids for the RVDs 11-20 + destination vector pFUS_A30B<br>3. Pick plasmids 21 up to N-1 + destination vector pFUS_B#N-1<br>Note: pFUS_B plasmids are labeled B1-B10 but they are used for RVDs 11-30 – if the RVD #N-1 is 19 or 29, use the same destination vector pFUS_B9)<br>4. Mix golden gate reaction #1 – for each set of vectors separately:<br>1-10 + pFUS_A;<br>11-(N-1) + pFUS_B(N-1)<br>or<br>1-10 + pFUSA30A<br>11-20 + pFUSA30B<br>21-(N-1) + pFUS_B(N-1)<br>a) 150 ng of each module vector + 150 ng of pFUS vector.<br>b) 1μl BsaI<br>c) 1μl Quick ligase or T-4 DNA ligase (QL is higher efficiency, but T-4 is much cheaper and works fine). 

<br>d) 2μl 10X T4 DNA ligase buffer (to final concentration of 1X) 

Note: If T7 ligase is used, add 2 ul of NEB Buffer 4 and 1 ul of 10 mM ATP<br>e) H2O up to 20μl total reaction volume 

<br><br> 

Note: Published protocol indicates using 20μL reactions, but it was found that 10μL reactions are reliably effective (same concentrations as in published protocol). Use 1⁄2 reactions (same concentrations, only 10μL total volume) can save some reagent. If a particular cloning reaction is somewhat difficult (failed more than once), it may be useful to use a 20μL reaction.<br> 

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5. Run cycle: 

10 cycles of (37 ̊C for 5min + 16 ̊C for 10min) 

then 50 ̊C for 5min

and 80 ̊C for 5min <br> 

With this cycle you will get hundreds of white colonies with 90-100% efficiency.

you can also use 20 oC instead of 16 oC<br><br> 

6. Plasmid-Safe nuclease treatment: this destroys all unligated linear dsDNA fragments including incomplete ligation products with lower number of repeats<br>fused; and cut and linearized vectors. The incomplete, shorter fragments would be cloned into the destination vector in vivo by recombination in the bacterial cell, if not removed (the start of the first repeat and the end of the last repeat are in the destination vector backbone, so the backbone has homology to each repeat module as they differ only in RVDs) <br> 

<br>To each of your golden gate #1 reactions add:<br>a) 1ml 10mM ATP<br>b) 1ml Plasmid-Safe nucleaseDay3&lt;br&gt;<br><br>Mini prep Miniprep the plasmids: Use enzymes AflII and XbaI to digest&lt;br&gt;<br><br>Incubate at 37 ̊C for 1h 

<br>Note: The Plasmid-Safe nuclease manual says you should inactivate the enzyme by heating the reaction to 70 ̊C for 30 minutes, but our experience, for bacterial transformatin, inactivation is not necessary<br> 

<br>7. Transform your chemically competent DH5alpha, Top10, or MACH with 10 ul reaction.<br> 

<br>8. Plate on Spec50 plates + 40μL of 20mg/mL X-gal +40μL of 0.1M IPTG. 

If STBL3 is used do not add X-Gal and IPTG as STBL3 can not be used for blue white screening. 

When plating transformations of the pFUSB vectors that have fewer repeats (especially less than 6 repeats), be careful to not plate all the cells as the efficiency is so high you can’t pick single colonies on day 2.<br><br>