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)
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(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 |
Take a p20, set it to 10uL and then pipet up and down.
THERMOCYCLER:
(Protocol EBGG)
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