DNA Purification Service
Interested parties can contact assistant core director to discuss preliminary approach of intended creation of a mouse model. Primary requirement to generate a mouse model at BCH using mouse core services is the PI must have an approved IACUC and IBC protocol for the desired model. After approval is granted the mouse core will co-ordinate with PI or lab member to submit request form and provide required reagents before scheduling an experiment.
Requirements
- Approved IACUC and IBC protocol for the desired mouse model.
- Submit electronic request form (eForm) for services signed by PI and designated lab member.
- Mouse core will co-ordinate with PI or lab member to collect required reagents before scheduling an experiment.
- A fund number or purchase order number to reimburse fees for requested services.
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Current Services
Core provide DNA purification services in following categories:
Plasmid DNA with a transgenic construct
Plasmid DNA is purified from fresh overnight bacterial culture using Qiagen Maxi Plasmid preparation (Endo-Toxin Free) kit. Plasmid DNA is digested with restriction enzyme/s to release transgenic construct from plasmid backbone. To ascertain complete digestion, ~100ng of digested DNA analyzed on Agarose gel along with appropriate DNA molecular weight ladder. After confirmation transgenic DNA is purified using Seakem GTG endonuclease free Agarose gel. Transgenic DNA is then isolated from agarose gel by using Qiagen gel extraction kit followed by ethanol precipitation. DNA is dissolved in TRIS buffer and used for microinjection.
Transgenic BAC DNA construct
BAC DNA is purified from fresh overnight bacterial culture using Qiagen Maxi Plasmid preparation (Endo-Toxin Free) kit. BAC DNA is digested with appropriate enzyme/s. To ascertain complete digestion, ~100ng of digested DNA is analyzed on Agarose gel and separated using BioRad Pulse field gel electrophoresis (PFGE) apparatus. After confirmation digested BAC DNA is fractionated using Sepharose CL4B column. Each fraction was analyzed on Agarose gel and fraction with BAC DNA is used for microinjection.
Tail biopsy DNA
Tail snips from pups at P7 is isolated using lysis buffer and Proteinase-K at 55oC overnight. DNA is precipitated and washed with 70% ethanol and dissolved in sterile water or TE buffer.
ES cell DNA
DNA is isolated from 96 wells to 6 wells. Tissue culture plate. ES cells are cultured without feeder cells for 1-2 passages, confluent wells are washed with PBS followed by lysis using a mixture of buffer and Proteinase-K at 55oC overnight. DNA is precipitated and washed with 70% ethanol and dissolved in sterile water or TE buffer.