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m (Some material we have available for you)
m (Standard PCR setup)
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| 10 μM primer A
| 10 μM primer A
| 0.8 μL
| 0.8 μL
| 8.0 μM
| 6.4 μL
| 10 μM primer B
| 10 μM primer B
| 0.8 μL
| 0.8 μL
| 8.0 μM
| 6.4 μL
| dd H<sub>2</sub>O
| dd H<sub>2</sub>O

Revision as of 11:37, 20 August 2012

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

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Course materials

This lab course is designed to provide a authentic research experience in recombinant DNA technology. Therefore you, the student, will have to prepare media and solutions you need to carry out your research by yourself. All basic chemicals you need to do so will be provided but in general you have to prepare your own solutions, buffers and media. You have to make them yourself in appropriate amounts sufficient to carry out your experiments. This is part of the course design and you ability to look ahead will be part of your assessment. Some good tutorials are listed on the lab website. For all additional procedures (for example tissue specific DNA extraction procedures), you need to consult the instructor to go over the procedures you are planning to use. Don't forget to do this well in advance so that there is enough time for the instructor to order the materials you need. In any case and for all your experiments you need to check if you have all your buffers and solutions available at the latest one session before you start your planned experiments! Please think ahead and let the instructor know IN ADVANCE 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). The instructor cannot provide special solutions or buffers, or sterile media or Agar plates with appropriate antibiotics ad hoc. Nobody will prepare them for you! This is your responsibility.

Since each project will progress in its own pace it is impossible for the instructor know in advance about all your needs. Therefore, ask if you are not sure. But do not assume the instructor can step in if you forgot to plan accordingly. You need to plan each step carefully to prevent unnecessary delays. Your careful planning is part of the course and will be evaluated. So use lag times to go over your procedures. Think one week ahead!. Otherwise you will loose important lab time and this will effect your performance and your grade. I would strongly suggest to generate a "needed items" list and put it into your notebook so that you know what to you need to take care of before the next lab session comes up. Please talk to the instructor frequently and as early as possible about your needs. This will help ensure that you can make effective use of your lab time.

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-ionized 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:
  • 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.

Agarose gel electrophoresis

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 0.5-4.0 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 6 reactions (+ the negative control and 1 spare). Please note that the positive control must be assembled separately since it receives different primers:

component for 1 reaction 8X premix
2X PCR master mix 10 μL 80 μL
10 μM primer A 0.8 μL 6.4 μL
10 μM primer B 0.8 μL 6.4 μL
dd H2O 7.9 μL / 4.4 μL 63.2 μL / 35.2 μL
final volume of premix 19.5 μL / 16 μL 156 μL / 128 μL
template DNA NONE - leave room for:0.5 μL diluted plasmid / 4.0 μL plant DNA NONE - can't go into premix. Why?

After you assembled the premix (on ice), you should have 156 μL (or 128 μL) in the tube. Divide this by 8 and pipette 19.5 μL (or 16 μL) into each of your 8 PCR tubes. Actually, you can trash the last one because it was the spare reaction anyway (to make for pipetting errors). Add 0.5 μL (4.0 μL) of your template DNA and don't forget to assemble the single positive control (plasmid and different primers!). If you programmed the thermocycler before you started assembling the reactions (did you?), you are now ready to go.

I want to see the above tables (both) in your notebook EACH TIME you setup a PCR reaction! Just scale the volumes up or down if you have different numbers of PCR reactions or larger volumes (max. is 50 μL per tube) to run.

Useful protocols (from OWW)

DNA protocols

PCR techniques

E. coli protocols

Hop DNA Isolation

Plasmid miniprep

Site-directed mutagenesis

Useful analysis tools

NCBI (The Center of Biotechnology Information)

NCBI Tutorials

IDT's SciTools collection

Fermentas Reviewer tool for restriction digest analysis

NEB cutter

Comprehensive Sequence Manipulation Suite

NCBI's ORF finder

Personal tools