General Comments File

Notes

1. This oligonucleotide will change the sequence of the m4b-CMV construct (Nucleotide Positions 3938-3943) from 5'-AGT ACT-3'(the ScaI recognition sequence) to 5'-AAT ACT-3'. This is achieved by using a primer that complements the sense strand to change the anti-sense strand sequence to 5'-AGT ATT-3'

2. Alternatively, you can use T4 DNA ligase buffer. T4 Polynucleotide Kinase (PNK) has 100% activity in T4 DNA ligase buffer, which contains 10mM ATP in the 10X stock. The PNK buffer does not contain ATP. Add 10 mM ATP for phosphorylation reactions and [³²P] ATP for radiolabeling the oligonucleotide.

3. These are trans oligos. One will remove the StuI site that is near the coding sequence (about 8 bp upstream) and generate a ScaI site in its place (Stu>Sca). The other oligo will remove the ScaI site and create a StuI site in its place (Sca>Stu).
4.