Miniprep Kit Using a Microcentrifuge

Aim

To familiarise us to the technique of extracting plasmid DNA from E. coli cells by using a Minipreb kit from Qiagene.

Protocol

The Miniprep Kit is used to extract plasmid, which is the first thing that we did.

1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times.
3. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
4. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
5. Apply the supernatant to the QIAprep spin column by decanting or pipetting.
6. Centrifuge for 30-60s. Discard the flow-through.
7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s. Discard the flow-through.
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60s.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
10. To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 μl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Nanodrop

Aims

To allow us to accurately measure the purity of DNA that have been extracted from E. coli using the Midiprep method.

Protocol

Figure 1: Steven trying out the Nanodrop machine

1. Select Nanodrop program from the desktop
2. To clean Nanodrop, add a drop of water on the spectrometer and press blank
3. After cleaning, wipe the water off
4. To equalize the equipment, add 3 μl of the buffer used in the sample and press blank
5. Wipe the buffer off
6. To measure sample, add 3 μl of the sample and press measure
7. If dealing with multiple samples, clean the equipment with water at regular intervals
8. After measurement, clean the equipment with a drop of water on the spectrometer and press blank

DNA Restriction Digestion

Aims

DNA digestion is a technique that allow us to cut DNA at specific sites. Therefore by running the digested plasmid or DNA on an agarose gel, we will be able to correctly see the size of the DNA.

Protocol

Digest was done by using EcoRI and PstI as the prefix and suffix of the BBa_04450 biobrick.

1. 20 μl solution should be made in total: 15 μl plasmid (with GFP/RFP), 1 μl EcoRI, 1 μl PstI, 2 μl Buffer (10x), 1 μl water. NOTE: Digestive enzymes should never exceed 10% of the total volume.
2. Add the reagents in this order: plasmid, water, buffer, enzymes.
3. Centrifuge for a few seconds to make sure that the mixture is at the bottom.
4. Heat block for 2 hours at 37 degrees C.

Gel electrophoresis

Aims

Gel electrophoresis allow us to separate DNA, RNA, or protein molecules by molecular weight using an electric field applied to a gel matrix after the restriction digestion step.

Protocol

1. Make up 1% agarose gel (1g of agarose in 100 ml of TAE buffer) and transfer 60 ml of molten agarose gel into the gel tray with 3 μl of Safeview
2. Wait for 30 min to allow the gel to harden
3. Transfer harden gel into the gel tank and add TAE buffer until the gel is completely submerge
4. Depending on the nature of the sample, 3μl of GeneRuler™ 1kb Plus DNA Ladder was used for analysing DNA
5. Loading buffer was then added together with the sample before loading onto the gel matrix
6. Run gel at 90V until separation is achieved and visualize using the gelDoc