840:153g:Materials

840:153g: Recombinant DNA Te(a)chniques

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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: we do have glycerol stocks of pSB1A3 and pSB1A7 (standard biobrick plasmids). You can also use part [BBa_I51020] or pBLUESCRIPT (which is not biobrick compatible). Before you use any plasmid for cloning, please verify the integrity by restriction digest with appropriate enzymes (should generate an informative pattern) AND PCR (VF2/VR primers for biobrick parts, M13 forward and reverse primers for pBLUESCRIPT). Be aware that BBa_I51020 has to be transformed into competent DB3.1 cells (because of the ccdB gene), whereas pBLUESCRIPT should be transformed into DH5α cells.
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 one of the following two DNA molecular markers from Fermentas. Select the one that best fits the size of the expected products. And please use both sparingly (1 marker lane for every 7 sample lanes) - 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.

Standard PCR setup

component	for 20 μL reaction	final concentration
2X PCR master mix	10 μL	1X
10 μM primer A	0.8 μL	0.2 μM
10 μM primer B	0.8 μL	0.2 μM
dd H₂O	to 20 μL
template DNA	0.2-4.0 μL	1-10 ng

Set up premix including reagents common to all reactions (i.e. everything except template; in other cases everything except primers). Prepare premix on ice for additional 10% reactions to have enough for all reactions planned. After adding last component (template DNA), start reaction immediately. ALWAYS include control with primers but no template DNA (negative control) AND control with plasmid DNA if you amplify from self extracted plant DNA. Use a table like the following for a setup of 5 reactions (+ the two controls, + 1 spare):

component	for 1 reaction	for 8 reactions
2X PCR master mix	10 μL	80 μL
10 μM primer A	0.8 μL	8.0 μM
10 μM primer B	0.8 μL	8.0 μM
dd H₂O	to 20 μL	to 200 μL
final volume of premix	19.5 μL / 16 μL	156 μL / 128 μL	template DNA
leave room for 0.5 μL diluted plasmid or 4.0 μL plant DNA	NONE - can't go into premix because of controls