50 ng of each piece of DNA being joined
(Use nanodrop to find concentration in ng/ul, then divide 50 by that concentration to find the required volume of DNA:
Conc: x ng/uL
Vol: 50/x uL)
x1 uL of DNA1
x2 uL of DNA2
...
y uL (100ng) Donor
2ul 10X T4 Ligase Buffer
2ul 10X BSA
1ul BsaI (enzyme)
1ul T4 Ligase (enzyme)
fill to 20uL with SDIH20 (put water in before the buffer and enzymes)
-------------
20ul total
(NOTE: Make sure that Buffer and Enzyme added last, enzyme after buffer)
| Add | Conc | Volm | Order of adding |
| DNA1 | c1 ng/ul | x1 = 50/c1 ul (50ng) | 2 |
| DNA2 | c2 ng/ul | x2 = 50/c2 ul (50ng) | 2 |
| GGDonr | c3 ng/ul | y = 50/c3 ul (100ng) | 2 |
| 10x T4 Ligase Buffer | 2ul | 3 | |
| BSA | 2ul | 3 | |
| BsaI (enzyme) | 1ul | 4 | |
| T4 Ligase (enzyme) | 1ul | 3 | |
| H20 | (20 - x1 - x2) ul | 1 | |
| Total | 20ul |
Take a p20, set it to 10uL and then pipet up and down.
Thermocycler:
protocol EBGG
37C for 5min
50X:
37C for 2.5min
4C for 0.5min
16C for 5.5min
37C for 10 min
80C for 20 min
4C hold
takes 8+ hours
(check protocol by looking up the paper or other online GG protocols)