Revision as of 09:51, 20 June 2010

Miniprep Kit Using a Microcentrifuge

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

Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times.
Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
Apply the supernatant to the QIAprep spin column by decanting or pipetting.
Centrifuge for 30-60s. Discard the flow-through.
Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s. Discard the flow-through.
Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60s.

Discard the flow-through, and centrifuge for an additional 1 min to remove residual wasg bufffer.

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 can be used to measure the DNA, RNA and protein

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

Gel electrophoresis can used to separate DNA, RNA, or protein molecules by molecular weight using an electric field applied to a gel matrix

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
Wait for 30 min to allow the gel to harden
Transfer harden gel into the gel tank and add TAE buffer until the gel is completely submerge
Depending on the nature of the sample, 3μl of GeneRuler™ 1kb Plus DNA Ladder was used for analysing DNA
Loading buffer was then added together with the sample before loading onto the gel matrix
Run gel at 90V until separation is achieved and visualize using the gelDoc

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+=Miniprep Kit Using a Microcentrifuge=
+The Miniprep Kit is used to extract plasmid, which is the first thing that we did.
+# Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
+# Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times.
+# Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
+# Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
+# Apply the supernatant to the QIAprep spin column by decanting or pipetting.
+# Centrifuge for 30-60s. Discard the flow-through.
+# Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s. Discard the flow-through.
+# Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60s.
+Discard the flow-through, and centrifuge for an additional 1 min to remove residual wasg bufffer.
+# 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 protocol=