Genomics

A quick note about quantitative real-time PCR.

In recent years there has been a push to standardize nomenclature and methods to allow reproduction of experiments.

From this desire, we get MIQE guidelines which is Minimum Information for Publication of Quantitative Real-Time PCR Experiments.

A growing number of journals are adopting these guidelines and it is worth considering when you are at the design phase.

The initial publication may be found here and a presentation (1hr) explaining it all here.

 

qPCR

Our ABI 7900ht qPCR machine is configured with 384 well sample block. This cannot be changed by users so it is impossible to run 96 well plate.

You don’t have to fill the 384 wells but the block requires that density plate

The machine on the right side of the room is Real time #2

Realtime #2 is currently the preferred machine for custom (longer than 2hr protocols).

Please note the reservation blocks are 30 minutes to allow both reservations for analysis and

custom runs (>2hrs)

ABI 7900ht real time PCR machines user’s guide and Primer express 3.0 manual.

RULES AND SOPS

This system is NOT on the network and data must be removed via USB drives

Real Time #2 is a Windows 7 system where everyone simply logs into to SDS user account (no pwd)

All data should be collected onto the D: drive, the User data folder specifically.

You are responsible for removing your old data from the system.  Failure to do so could result in machine failure.

DO NOT EVER turn off the 7900. it must remain on always

RT#2 uses SDS 2.4 for acquisition.  There is a version of the software for off-line analysis that you can install on lab PC’s for data analysis and plate layout (SDS 2.4).

I also have Primer Express 3.0 ( ABI primer design software)  if anyone needs it.***Important note: Primer express will not run on a dual core machine (all new pcs) and neither software will run on Mac. I have been able to get the software to run on Win7 64bit

Be considerate of other users.  Do not sign up for time slots you don’t need.

Start on time.  Runs take 2 hours and if you start your run late it will snowball throughout the day.

IMPORTANT: If you plate is running into someone’s time slot, They have the right to abort the run a dispose of your plate! So if you are running late check the schedule before starting, and notify any effected parties.

Do not write on your plates or set them on dirty counters.  Any filth on the bottom of the plate gets baked on the block resulting in poor performance.

Any concerns or problems, let Todd know

 NOTE: The plate centrifuge has died in an irreparable manner.

It has been replaced with the Lab Hack Plate spinner.  More info here

Agilent 2100 BioAnalyzer

On the first bench along the east wall, is the BioA. The Bioanalyzer is a chip based electrophoresis platform that allows analysis of RNA,DNA or protein (depending on the chip used).

https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-dna-kits-reagents/bioanalyzer-dna-analysis-228248

This is an excellent BioA overview

https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument/2100-bioanalyzer-instrument-228250

the most common use is to evaluate RIN score prior to sequencing RNA (.overview-rna-quality-automated-electrophoresis-5994-7327EN-agilent)

NanoDrop One C-

We have a NanoDrop one with cuvette capability. There are currently no charges for use. It is imperative that users follow the posted rules for shutting the system down, cleaning the pedestal, and proper disposal of tips and waste.

Turner 20/20N –

Tube-based luminometer with dispensers .

 

Imaging workflows in 632

  • Our fluorescent plate reader is available on the scheduler as Victor 3

This can read fluorescence or luminescence in multiwell plate formats from above or below. It has conventional epifluorescence filters for GFP and RFP as well as DAPI.  This system is currently not charged but all use must be recorded.

  • Cellometer Vision- a slide-based automated cell counter
  • Anthos-96 well absorbance plate reader with filters for A590. A550 A405;A340nm*

*currently the 340nm lamp is reading low energy so a different UV spec would be a better option

Here is a quick overview of how to use the Anthos (anthos 1.mp4)

  • Cary UV 50- Absorbance based spectrophotometer
    • Please be kind to this elder statesman

Genomics Billing

The ABI systems are under contract and users split the cost proportionally. The last rate was $14.00 per run

The GenePix system has no contract and users split the costs of repairs. This system is past end of life with parts not being available. I would not plan on seeing this much longer