Rules for the design of compatible overhangs (taken from this paper, see page 6 of the PDF = page 146 of the book)
- Avoid using the same overhang sequence twice
- Avoid palindromic overhangs
- Avoid having overhangs that share 3-4 consecutive nucleotides in their sequences (results in reduced efficiency)
(An example excel file is attached)
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
NOTE: If GGDonr is too concentrated, dilute it with EB or water.
NOTE: Ligase buffer does not like to be freeze-thawed, so use one-time-use aliquots.
x1 uL of DNA1
x2 uL of DNA2
y uL (100ng) Donor
2ul 10X T4 Ligase Buffer
2ul 10X BSA
1ul BsaI (enzyme) HC (high concentration)
1ul T4 Ligase (enzyme) HC (high concentration)
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)
Take a p20, set it to 10uL and then pipet up and down.
ADD | CONC. | VOLUME | ORDER |
| DNA1 | c1 ng/ul | x1 = 50/c1 ul (50ng) | 2 |
| ... | ... | ... | ... |
| DNAn | cn ng/ul | xn = 50/cn ul (50ng) | 2 |
| GGDonr | d ng/ul | y = 50/d ul (100ng) | 2 |
| 10x T4 Ligase Buffer | 2ul | 3 | |
| 10x BSA | 2ul | 3 | |
| BsaI (enzyme) | 1ul | 4 | |
| T4 Ligase (enzyme) | 1ul | 3 | |
| H20 | 20 - (x1 + ... + xn + y) ul | 1 | |
| TOTAL | 20ul |
THERMOCYCLER:
(Protocol EBGG)
37C for 5min
Part 1
50X:
37C for 2.5min
4C for 0.5min
16C for 5.5min
Part 2
37C for 10 min
80C for 20 min
4C hold (for 8+ hours)
(Check protocol by looking up the paper or other online GG protocols)