Contents
Reagents needed and providers:
| Reagents | ||||
| Tris Hcl pH 7.5 | VWR | A4263.0500 | £21.90 | 500ml |
| PEG 8000 | Sigma | P1458-25ML | £15.63 | 25ml |
| MgCl2 | Thermofisher | AM9530G | £29.70 | 100ml |
| DTT | VWR | A3668.0050 | £30.60 | 50ml |
| NAD | NEB | B9007S | £16.80 | 0.2ml |
| dNTP | Thermofisher | 15363397 | £49.50 | 1ml |
| Phusion | NEB | M0530S | £56.00 | 100U |
| Taq Ligase | NEB | M0208S | £49.60 | 2000U |
| T5 exonuclease | NEB | M0363S | £41.60 | 1000U |
| Water | VWR | MDTH46-000-CI | £33.00 | 6x100ml |
2X ISO Reaction buffer
| 2X ISO Reaction buffer | ||||
| Stock [] | Final [] | |||
| Tris Hcl pH 7.5 | 1M | 200mM | 220 | 1000 |
| PEG 8000 | 40.00% | 10.00% | 275 | 1250 |
| MgCl2 | 1M | 20mM | 22 | 100 |
| DTT | 1M | 20mM | 22 | 100 |
| NAD | 50mM | 2mM | 44 | 200 |
| dNTP | 10mM | 0.4mM | 44 | 200 |
| H20 | 473 | 2150 | ||
| total (ul) | 1100 | 5000 | ||
| Reactions # | 104 | 474 | ||
All measurements are in ul. Make 133 ul aliquots and store at -20C. Good for one year. 1100 makes 8 aliquots, equivalent to about 104 final gibson reactions. Scale up using the attached XLS file (recipe.xls).
Master mix
| Master mix | ||||
| Stock [] | Final [] | |||
| 2X ISO React. buff. | 133.2 | 666 | ||
| Phusion | 2U/ul | 0.033U/ul | 3.3 | 16.5 |
| Taq Ligase | 40U/ul | 5.3U/ul | 26.6 | 133 |
| T5 exonuclease* | 5U/ul | 0.005U/ul | 0.2 | 1 |
| H20 | 36.7 | 183.5 | ||
| total (ul) | 200 | 1000 | ||
| Reactions # | 13 | 66 |
Store at -20C. Good for 2 years. All measurements are in ul.
*T5 comes from NEB at 10U/ul. Make 1:2 dilution in IX T5 buffer (NEB #4) to create an intermediate stock solution
Credits and other protocols
There are several protocols for Gibson recipes on the internet. The one above is taken from the Kimble lab at UW-Madison (GibsonCloning.pdf). They usually work at ratio of 15ul of MIX to 5ul of DNA. The only one I found that works at 1:1 is the one by Ethan Ford (2x-gibson-assembly-master-mix.pdf)