---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2010-07-03 04:42:26+00:00
layout: post
slug: restriction-digests-various-gigas-gdnas-of-macs
title: Restriction Digests - Various gigas gDNAs of Mac's
categories:
  - 2010
  - Miscellaneous
tags:
  - 5-azacitidine
  - Crassostrea gigas
  - DNA Quantification
  - EtOH precipitation
  - gDNA
  - HpaII
  - larvae
  - MspI
  - NanoDrop1000
  - Pacific oyster
  - restriction digestion
---

Performed restriction digests. Made dilutions of all DNAs involved of 25ng/uL. Made enough for a total of 9 digests could be performed on each DNA. This allowed using 10uL of each DNA for each rxn, more mileage out of the lowest concentration sample (R37-01), and allowed for the use of master mixes when preparing the digests. [All calculations/dilutions/master mixes can be seen here](https://spreadsheets.google.com/ccc?key=0AmS_90rPaQMzdGdTZUJBQW8yT0ZjMkF3QzZ4U2dMNEE&hl=en&authkey=CN7umpIO). Each DNA was digested individually with HpaII, MspI and undigested. Incubated the digests 4hrs @ 37C. After digestion, performed an EtOH precipitation. Added 0.1 vols of 3M NaOAc (pH=5.2), then 2.5 vols of 100% EtOH. Mixed by inversion and incubated 30mins @ -20C. Pelleted DNA 16,000g, 30mins @ 4C. Discarded supe. Washed pellets with 1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Discarded supe. Resuspended DNA in 10uL PCR H2O and spec'd.

Results:

![](https://eagle.fish.washington.edu/Arabidopsis/20100702%20gDNA%20digests%20ODs.JPG)

Well, the recovery of DNA is very low. The best recovery is ~50% while the worst is around ~1%.

I did not proceed with the intended qPCR due to the low yields and the fact that I don't know if we've previously tested how sensitive our assay(s) our for our target genes. Will discuss with Steven/Mac next week.
