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Cleaning up RE digest results - advice wanted (with pictures)

RE digest

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#1 SteveB

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Posted 08 June 2022 - 07:42 PM

I am a Scientific lab tech at a high school conducting a number of Biotech experiments. As part of the yr 12 Biology genetics and Biotech unit, we run a DNA digest over a couple of lessons. The results aren't bad but can be variable, and I would like some advice on how to improve the results.

 

Protocol as follows:

λ bacteriophage DNA: ~1ug (3ul)

Buffer : 2ul (10x buffer)

Enzymes : 2 units (2ul) (This is using a dilution of the 12 unit/ul stock supplied from the biotech company into a 1 unit/ul aliquot, which is easier for the students to pipette. The dilution is only made just before each experiment

Water : 13ul (autoclaved RO/DI water, aliquoted in sterile conditions)

- Incubated at 37˚C for 1 hour. I then add 2ul 10X loading dye to stop the reaction, then immediately freeze.

 

The next class, all tubes (& pre-cut lambda with EcoRI + HindIII) are placed into the water bath at 65˚C for 2 minutes to 'unstick'. Gel is 0.9% agarose with Redsafe dye. 10ul per lane (they make 2 lanes for each tube). Gel is run for 35 minutes at 100V. Gels are then placed on a UV illuminator to take photos.

 

Results:

Lane 1- Control (no enzyme)

2- BamHI

3- EcoRI

4- HindIII

5- MWM (pre-cut Lambda with EcoRI + HindIII) 6- 1kb DNA ladder

 

These are some average photos, some are worse or better. Obviously common students mistakes, poor pipetting, or just forgetting components affect the results. Is there anything else that can be done to help improve these.

REdigest3.jpg REdigest1 (1) - .jpg

Much appreciated.


Edited by SteveB, 08 June 2022 - 08:26 PM.


#2 mdfenko

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Posted 09 June 2022 - 10:04 AM

This may not help with all of the problems but…

 

some lanes appear a little overloaded

 

some were left sitting in the well for an extended period before starting the run (allows the sample to migrate into sides)

 

some either well contained bits of gel (from when comb was removed, need to flush well before loading) or pipet tip broke a chunk of gel while loading (need steady hand when loading)

 

how long do the gels sit before placing on the transilluminator? Diffusion will continue and cause bands to get fuzzy


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#3 SteveB

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Posted 14 June 2022 - 08:02 PM

Actually that's quite helpful, thank you.

 

The time it sits in the tank can be fairly variable and not something I had considered to affect the results so much.

The students pipetting into the wells can take a while, so from the first well to the last well (and then starting the power pack) is certainly enough time to start diffusing material out into the gel. Secondly, due to the amount of students or if I am doing another experiment, I may leave the gels in the tanks for a short period before getting them out to take a photo. This of course leaves more time for the DNA to diffuse out from when the run finished.

 

I will keep this in mind and make sure they are removed and imaged as a priority, and see if that improves things.






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