General Molecular Biology Lab Tips and Practices

Pipetting

Hold the container of liquid to be dispensed in the left hand and view from the side. Adjust the pipet to the desired volume. Depress the pipet handle down to the first stop and then put the tip in the top of the liquid to be dispensed. Release the handle slowly, watching the liquid rise into the tip. As the pipet is withdrawn from the container, wipe off any material that is adhering to the outside of the tip, as it may amount to several µL in volume and may throw off measurements of small quantities. Place the pipet end in the top of the liquid (if any) in the new container and slowly depress the handle to the first stop. Withdraw the pipet from the second container and release the handle.

Be sure to use the correct size tip with a pipet. P1000 µL pipets use a larger tip than P200, P20 or P10. We used Pipetman brand. Filter tips are available for solutions where cross-contamination is especially problematic (e.g. RNA and PCR solutions). These tips cannot be autoclaved and come presterilized and wrapped.

Change pipet tips after every dispensation.

Spin down small quantities of solutions in a nanofuge before and after dispensation to concentrate all the liquid at the bottom of the microfuge tube.

Things not to do:

1. Don't release the plunger handle while the tip is in the second container, as you will suck the liquid back up into the pipet.
2. Don't dispense liquid onto the side of the tube, as it may be hard to get off.
3. Don't put the plunger down to the final stop and cause a bubble to be omitted, as you increase the chances of cross-contaminating solutions.
4. Don't stick the tip further into the container than necessary, as you increase the amount of material adhering to the outside of the tip.
5. As pipet tips are not angled, it is possible to close them off by pressing them too hard against the bottom of a container.
6. Pipet tips come in large plastic bags. They should be loaded into plastic tip boxes and autoclaved at 120C before use.

Handling RNA

Single-stranded RNAs are particularly susceptible to degradation from the 5' end and must be handled with special care. Some precautions to take:

1. Use a new box of tips for RNA. Keep the box of tips closed when not in use. Keep microfuge tubes closed when not dispensing.
2. Replace gloves if they come in contact with skin or hair.
3. Keep RNA tubes on ice at all times when not in use.

Gel electrophoresis

Thin agarose gels should be used for quantification as they transmit more light.

Denature lambda gel markers at 65C for 3-5 minutes before using, as otherwise they form supercoiled plasmid structures that run more quickly in a gel.

Keep the level of the electrophoresis buffer solution shallow in order to improve visibility during loading of a gel. Move pipet tip downwards towards gel until it can be seen to move just a bit. Overinsertion of the pipet tip can result in a hole in the gel. Underinsertion of the pipet tip causes most of the solution to diffuse sideways without entering the well.

Agarose solution can be stored in a 55°C oven until ready for use. The solution should be poured quickly as otherwise lumps will cause the gel to be cloudy.

With a dense agarose gel (3-5%), a 50 bp fragment can be separated from a 100 bp fragment from a 150 bp fragment. For tough separations use the Metaphor gel from Bio-Whittaker. Polyacrylimide gels give much better results for high-resolution measurements.

In a gel, lower MW fragments bind less ethydium so the fluorescence signal isn't proportional to concentration. In contrast, the darkness of a lumigram is a linear response.

General

Place tubes in the centrifuge with the hinge pointing outward radially, as then any pellets stuck to the tube interior will be located directly below the hinge.

Biohazard trash can be autoclaved at 120°C and then discarded with regular trash.

The quality of water is very important for molecular biology both in terms of ionic content and biological contamination. Distilled, deionized water should be good enough. Running "no template" controls on all the experiments is an important check of water contamination.

Enzymes are very unstable. Try to hold tubes of enzymes so as not to warm them excessively with your hands.

4C is a good temperature to hold DNA and other reagents overnight. For long-term storage, add glycerol and freeze at -20C.

Ligation is the step in a molecular biology protocol which is most likely to fail.