Isolating DNA and running an enzymatic reaction

For our first lab we created DNA ladders using pPSU1 and pPSU2 using the ZymoPure Plasmid Miniprep Kit. The Lab procudure can be found here.


plasmid: Small, typically circular, strand of DNA

restriction enzyme: Enzyme that cuts "cleaves" DNA at a specific sequence of bases.

sticky ends: Unpaired nuceotides at the end of a DNA strant. Also called "cohesive ends" and "overhangs". These sticky ends are useful because they ensure the cut DNA is in the correct direction when inserted into a plasmid.

supernatant: Liquid that separates from solid residue

copy number: Refers to the number of replications a plasmid will have in a host cell. Plasmids will be identified as low, mediumm or high copy number. Plasmids regulate themselves to ensure they do no overly burden the host cell.

Lab notes:

Following the procedure we collected the E. coli culture and buffers from the ZymoPure kit.

We took the E. coli samples that had the plasmids in them, suspended them, lysed them, neutralized and then washed with buffers.

This was my first time in a bio lab so Rahma kindly showed Rae and I how to pipette and what vortexing is. I was surprised to see how much technique was involved in pipetting to prevent drops of liquid sticking to the outside of the pipette tip, making sure all the liquids mixes at the bottom of the tube, not stuck on the wall as well as preventing any airbubbles.
By spinning a standard tube with the lysate most of the cellular mater collects at the bottom and we transfered the supernatant with a pipette. To "wash" the DNA it was put in the top of the spin column and most of the solution flowed through, but the DNA gets trapped in the silica membrane. ZymoPURE uses a few washes, then there is a substance, called DNase|(RNase-Free), that pulls the DNA out of the silica. This process of extracting one material from another by washing with a solvent is called elution. At this point we isolated the DNA.
To measure the concentration of DNA in the solution we used the Nanodrop spectrophotometer. This allowed us to dilute the solution to get the appropriate amount for the reaction.
Initial concentration [ng/uL] 1787 1814
Diluted and remeasured [ng/uL] 243 367
Amount needed for 1ug of DNA 4.1 uL 2.7 uL
We put the DNA into wells at one end of the gel and applied 120V of potential. As the DNA is negatively charged it migrates through the gel towards the positive end. The shorter molecules travel faster through the gel and thus bands of DNA separate out based on size (number of base pairs). This is called the banding pattern. The gel, agarose, has a dye in it, SYBR Safe, which binds to DNA and floresess under UV light, which is how we are able to see the banding patterns with a transilluminator.


Q. What software are you using to read the sequence files?
A. Benchling

Q. What are the distances between the PstI (CTGCAG) sites in each plasmid? What are the distances between the EcoRV (GATATC) sites in each plasmid?
A. pPSU1 has 7 Pstl (CTGCAG) cleavage sites and EcoRV has 5 (GATATC) cleavage sites.
Distance between cleavage sites in pPSU1 by Pstl: 494, 1994, 994, 694, 794, 894, 4094 [basepairs]
Distance between cleavage sites in pPSU1 by EcoRV: pPSU2 has 9 Pstl (CTGCAG) cleavage sites and EcoRV has 3 (GATATC) cleavage sites.
Distance between cleavage sites in pPSU2 by Pstl: 1494, 44, 94, 194, 294, 394, 494, 594, 4094
Distance between cleavage sites in pPSU2 by EcoRV: 744, 2994, 3994
These can be found by highlighting the distance between the genes on benching.

Q. Where do PstI and EcoRV cut within their recognition sequences?
A. The sticky ends created by Pst1 and EcoRV can be seen below. These were copied from Sigma-Aldrich

Q. What gene is encoded into the plasmids?
A. AmpR is in both pPSU1 and pPSU2

Q. How many copies of the plasmid would you roughly expect in each cell?
A. pPSU1 and pPSU2 are both high copy number, so ~500 copies per cell

Q. How do the plasmids' replication relate to that of pBR322 (genbank)?
A. pBR322 is a medium copy number, where as pPSU1 and pPSU2 are high copy numbers.