Plasmid Isolation

Colony #2 from 20120426

Inoculated 5mL of 1x LB + Amp (100ug/mL) with colony #2 based off the of the PCR screening on 20120426 in a 50mL conical tube. Incubated @ 37C, 200RPM O/N. Plasmid DNA was isolated using Qiagen’s Mini Prep Spin Kit. Plasmid was eluted with 50uL of Buffer EB and spec’d on the Roberts Lab NanoDrop 1000.

Results:

[Plasmid] = 288.5ng/uL

Looks good; will proceed with linearization.

Restriction Digestion

Performed a restriction digestion using HindIII (NEB) @ 37C for 2hrs and then heat inactivated for 20mins @ 65C. Recipe:

• Plasmid (1154ng): 4uL
• 10x Buffer 2: 5uL
• H2O: 40uL
• HindIII: 1uL

Ran half of the reaction on a 0.8% agarose low TAE gel, along with ~500ng of undigested plasmid.

Results:

Gel Loading:

Lane 1 – Hyperladder I (Bioline)

Lane 2 – Undigested construct

Lane 3 – HindIII digestion of construct

We see exactly what we want to see: the construct is fully linearized and runs at the expected size (~5400bp). It runs about half way between the 5000bp and 6000bp markers on the ladder. Will quantify and prepare a dilution curve for the RLP qPCR assay.

Restriction enzyme was selected based on what the construct should look like and HindIII is a single cutter in this instance. Below is an image of the construct, with the insert (AF133090) in green. Of nine common enzymes, four are indicated in the image below as being single cutters. HindIII was selected based on availability in the lab.