Amypasternack-022606
From FreeBio
Aim
To purify 1.1 kb PCR product by Dpn1 digestion and purification from a gel
To obtain sufficient DNA to perform recombination
Methods
- Dpn1 Digest
Added 1.5 uL of Dpn1 to thrBC::cat PCR product from 2/20/06 to digest methylated (template) DNA. Incubated at 37 C for 30 min. DNA amplified by PCR will not be affected.
- Gel Preparation (with Nile Blue)
1% agarose gel (1 g agarose, 100 mL TBE (1x)). Heated for 1 min in microwave, added 10 uL Nile Blue (1:10,000) while solution was hot but touchable.
Running buffer: 600 mL TBE (1x), 60 uL Nile Blue (1:10,000)
Loading dye (6x): 1 uL loading dye per 5 uL sample. 8 uL loading dye to 40 uL sample.
- Gel Electrophoresis
Loaded 2 log ladder (lane 1), small sample of PCR product (lane 2), rest of PCR product (wide lane 3), and 1 kb ladder (lane 4)
Ran gel, 130 V, 40 min.
- Visualization and Purification of DNA
Cut lanes 1 and 2 and post-stained with EtBr (1:10,000) 10 uL EtBr in 100 mL TBE.
Visualized band from lane 2 to approximate location of band from lane 3, cut band from lane 3 out of gel, post stained gel in EtBr to look for residual DNA at margins of cut.
Followed QiaGen Gel Extraction Protocol to purify DNA from .428 g gel fragment
Measured DNA concentration with NanoDrop.
