User:Mara Peterson/Notebook/Mara Peterson/2015/12/16

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Overview

Performing plasmid minipreps on the liquid cultures started yesterday, quantifying the DNA yield, performing restriction digests to verify presence of insert, and sending promising samples out for sequencing.

Plasmid Miniprep

Followed the Sigma kit instructions. Eluted in 50 µL nuclease-free H2O.

DNA Quantification

Sample ID 260/280 ng/µL
HA1_v0120 1 1.91 594.0
HA1_v0120 2 1.91 591.6
EM7:ZeoR_v0120 1 1.90 628.8
EM7:ZeoR_v0120 2 1.90 480.5
EM7:ZeoR_v0120 3 1.90 529.7
EM7:ZeoR_v0120 4 1.92 544.5

Restriction Digest

Using 1 µL (~500 ng) of each plasmid as the template.

Reagent Volume (µL)
Plasmid DNA 1
10x FD Buffer 2
EcoRI 1
PstI 1
H2O 15

Incubate at 37 °C for 5 minutes, then run on the gel, 100 V for 50 minutes.

Gel Results

Image:
File:2015-12-16 ZeoR and HA1 restriction digest and HPK-CFP PCR.tif

See first six sample lanes. Both HA1 samples appear to have the insert (~100 bp), and EM7:ZeoR samples 1 and 2 appear to have the insert (~500 bp). Will send those four samples to SOLS Core lab for sequencing using P0001 primer.