Molecular biology services

eagle-i ID

Resource Type

Properties

1. Fee for service

   Yes

2. Resource Description

   "<b>PCR, PCR clean up & Cloning</b> PCR of genomic DNA or BAC DNA is done using custom primers. Primers can also be designed at the facility. The optimization and the clean up of PCR products are done using various procedures. The cleaned up products can be directly processed for Sanger sequencing at the facility or used for cloning. <b>Transformation and Cloning</b> Design of cloning strategy followed by directional or non-directional clonings are performed using appropriate competent cells and vectors. Insertions sites are checked and orientations are determined by either restriction digestion and/or sequencing. PCR products are cloned using Invitrogen’s TA cloning system. <b>Sub-cloning</b> After designing the cloning strategy, DNA is sub-cloned into an appropriate expression vector followed by the preparation of the construct DNA for micro-injection into mouse zygotes. <b>Site-directed Mutagenesis</b> Mutagenesis including point mutation, insertion and deletion of single or multiple amino acids and large insertion are done using Stratagene’s Quick Change kit. This includes design and synthesis of the mutagenic primers, mutagenesis and verification by sequencing. The synthesis and the PAGE purification of the primers are outsourced to Invitrogen. <b>AMPure Bead Purification of Amplicons and DNA Fragments</b> Agencourt (Beckman) AMPure XP bead-based DNA purification of amplicons efficiently removes unincorporated dNTPs, primers, primer dimers, salts and other contaminants, resulting in high quality DNA suitable for sensitive downstream applications such as sequencing, genotyping, PCR, and sub-cloning. Recovery: 70 - 90% of double stranded and single stranded DNA with fragment sizes 150 bp - 10 kbp. This is the recommended purification method during sample preparation in most protocols for various new generation sequencing platforms."

3. Service Fee URL

   http://www.med.upenn.edu/genetics/dnaseq/pricing.shtml

4. Service Provided by

   Penn Genomics Analysis Core

5. Website(s)

   http://www.med.upenn.edu/genetics/dnaseq/molbio.shtml

6. Related Technique

   Site-directed mutagenesis

7. Related Technique

   Polymerase chain reaction

8. Related Technique

   Recombinant plasmid cloning