Figure
1. UPPERCASE - nucleotides from pBI121 or from the PCR primers lowercase - nucleotides from the adaptors NNNN - nucleotides from original primers XXXXXX, YYYYYY - recognition sequences for enzymes X and Y nnnnnn - arbitrarily chosen nucleotides for primers or adaptors Restriction cuts generating sticky ends are shown as red lines. |
A. Oligonucleotides BS1: 5'GATCCGGGCCCG3' BS2: 5'TCGACGGGCCCG3' |
B. How the oligonucleotides would pair
in-vitro 5'GATCCGGGCCCG3' 3' GCCCGGGCAGCT5' |
Additional criterion: design different internal duplex
sequences for each pair of oligos, such that oligonucleotides
from one pair will NOT form duplexes with oligos from the other
pair.
Creating your construct
in silico In the remaining steps, you will use UGENE to create your construct. One complicating factor is that programs that work with DNA sequences almost universally represent only a single strand, where the complementary strand is implied. For cloning steps, the representation of sticky ends is a special case. We will also end up typing in each adapter pair as if it was a single strand of a double-stranded duplex. These steps will be described in more detail as we proceed. To keep all of your files organized, start UGENE and create a new project called as4. |
Open pBI121.gen in UGENE, and do the following.
Again, check in the Details panel to make sure that you have added the correct sequences, before proceeding to the next step.
- Obviously, you need to make sure you are adding the sequence in the correct strand and orientation
- Set the position to insert to the end of the sequence
- Save to the same file, LepR3_modprimer.gb ie. just overwrite the file you created.