The DNA fragments are cut from the agarose gel with a scalpel. Three parts of QG buffer is added to one part of gel and the mixtures are dissolved at the temperature of 50°C. The tubes are vortexed every 2-3 minutes to help dissolve the gel. 1 part of isopropanol is added to the sample and mix. The microcubes were spinned in the microfuge for 1 minute.
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==Set up ligation==
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====QIAquick gel extraction microcentrifuge protocol====
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# Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
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# Weight the gel slice and add 3 volumes of buffer QG to 1 volume of gel (100 mg ~ 100 µl)
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# Incubate at 50°C and invert the tube gently at regular intrerval until the gel has completely dissolved
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# After the gel has dissovled completely, check that the color of the mixture is yellow
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# Add 1 gel volume of isopropanol to the sample and mix
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# Place a QIAquick spin column in a 2 ml collection tube
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# To bind DNA, apple the sample to the QIAquick column and centrifuge for 1 min
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# Discard the flow through and place the QIAquick column back into the same tube (Maximum volumn is 750ul)
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# Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min and discard the flow through and place the QIAquick column back into the same tube
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# To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min and place the QIAquick column back into the same tube
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# Centrifuge the column for a further 1 min
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# Transfer the column into a clean 1.5 ml micriocentrifuge tube
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# To elute DNA, add 30 µl of Buffer EB to the center of the QIAquick membrane and allow to stand for 1 min
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# Centrifuge the column for 1 min and transfer the eluate to a clean 1.5 ml micriocentrifuge tube
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====Weighing====
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All gel extractions weighed 0.3g.
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===Set up ligation===
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===Transformation protocol===
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==Transformation protocol==
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# Thaw a 200µl aliquot of the desired strain of ''E. coli'' and add the transforming DNA (10 ng of vector DNA in 10 µl). Incubate for 45 min at 42°C.
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Please refer to: [[Team:Newcastle/Transformation of E. coli|Transformation of ''E. coli'']] for materials required and protocol.
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# Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
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# Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
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# Plate out 100-200 µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
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# Incubate plates overnight at 37°C.
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===Nanodrop Protocol===
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Nanodrop can be used to measure the DNA, RNA and protein
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# Select Nanodrop program from the desktop
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==Nanodrop Protocol==
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# To clean Nanodrop, add a drop of water on the spectrometer and press blank
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Please refer to: [[TeamNewcastleNanoDrop Spectrophotometer| Nanodrop Spectrophotometer]] for materials required and protocol.
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# After cleaning, wipe the water off
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# To equalizen the equipment, add 3 μl of the buffer used in the sample and press blank
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# Wipe the buffer off
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# To measure sample, add 3 μl of the sample and press measure
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# If dealing with multiple samples, clean the equipment with water at regular intervals
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# After measurement, clean the equipment with a drop of water on the spectrometer and press blank
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{|
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|[[Image:Newcastle_nanodrop_1.jpeg|thumb]]
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|[[Image:Newcastle_nanodrop_1.jpeg|thumb|A typical curve to be achieved from a nanodrop spectrophotmeter]]
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|[[Image:Newcastle_nanodrop_2.jpeg|thumb]]
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|[[Image:Newcastle_nanodrop_2.jpeg|thumb|Phil analyzing a sample on the nanodrop machine]]
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|[[Image:Newcastle_nanodrop_3.jpeg|thumb]]
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|[[Image:Newcastle_nanodrop_3.jpeg|thumb|Analyzed data on the screen]]
The aim of today's Lab practice was to extract the plasmids for GFP and RFP, digest the 2 plasmids and extract the 2 inserts and 1 of the backbones (vector)from an agarose gel. From this a ligation was set up.
Materials and Protocol
QIagen Miniprep Kit Using a Microcentrifuge for Plasmid Extraction
Please refer to: Qiagen Minipreps for materials required and protocol.
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