840:153g:Materials

Course materials

This lab course is designed to provide a real life experience of work in a genetic engineering lab. Therefore, you will have to prepare project specific media and solutions yourself. Most of the chemicals you need will be available, but not all solutions and buffers will not be ready for you. You have to make them yourself if you are using special protocols (for example plant specific DNA or RNA extraction procedures). If you need something that you cannot find, ask the instructor or Larry in the biology stock room. You need to check if you have all your buffers and solutions available one session before you start your planned experiments! Please think ahead and let me know of any special needs you may have. If you don't plan for your next session, your work will very likely be delayed (which will affect your grade). I cannot provide special solutions or buffers, or sterile media or Agar plates with appropriate antibiotics ad hoc. And nobody will prepare them for you! Since each project will progress in its own pace it is impossible for me know in advance what you might need. Therefore, ask if you are not sure. But do not assume I can step in if you forgot to make something. I cannot. I need you to plan ahead to prevent unnecessary delays. Your careful planning is part of the course and will be evaluated. You also will learn to prepare basic solutions by yourself. This will become very important in case you end up working in a lab, either on the job or in grad school. Therefore, it is fully your own responsibility to plan ahead and prepare what you need for the next lab. Think ahead and read the next steps in the protocol. Otherwise you will loose important lab time and this will effect your performance (and your grade). I would strongly suggest to put a "needed items" list into your notebook so that you know what to you need to take care of before the next lab session. Please talk to me frequently and as early as possible about your needs so that I can help you find the materials (or alternative ways).

Some material we have available for you

• standard plasmids: pSB1A3 and pSB1A7 from the Registry have issues. Please use part BBa_I51020 and pBLUESCRIPT. If you isolate plasmids, please prepare both. We need pBLUESCRIPT as positive control in PCR. If BBa_I51020 works, we can use it for both, cloning and as PCR control.
• Plasmid isolation: Gene Jet Miniprep Kit from Fermentas
• Resctiction digest: FastDigest EcoR1, Pst1, Spe1, and Xba1 from Fermentas
• PCR: 2x PCR Master Mix (Choice Tag) from Denville
• Agarose gelectrophoreses: make 1X TBE buffer from 10X stocks by adding 500 ml 10X TBE buffer to 4500 ml water (de-inoized tap water)
• we use either of the following two DNA molecular markers from Fermentas (please use both sparingly - they are very expensive):
  • Fast ruler low range ladder (50-1500 bp) for PCR products
  • Gene ruler 100 bp ladder plus (100-3000 bp) for plasmid preparations:
• antibiotics (Amp, KAN, Tet, ...) come in 1000x stocks. That means, you must use 1 mL per L of medium!
• X-GAL and IPTG stocks are also 1000x concentrated

Make sure to store all enzymes (and master mixes containing enzymes) at -20°C (freezer compartment). These materials have to be kept on ice constantly while assembling reaction (keep those on ice until finished). They should be out of the freezer only as long as absolutely necessary. Otherwise your experiments may not work properly. Buffers, solutions and media are best stored in the fridge.

Useful protocols (from OWW)

DNA protocols

PCR techniques

E. coli protocols

Plant DNA isolation

Plasmid miniprep

Useful analysis tools

IDT's SciTools collection

Fermentas Reviewer tool for restriction digest analysis

NEB cutter

Comprehensive Sequence Manipulation Suite

NCBI's ORF finder