Team:Newcastle/Qiagen Minipreps
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===Materials=== | ===Materials=== | ||
- | [[Image:Newcastle_lab_6.jpeg|right]] | + | [[Image:Newcastle_lab_6.jpeg|350px|right]] |
# Scalpel | # Scalpel | ||
# Eppendorf tubes | # Eppendorf tubes |
Revision as of 10:20, 12 August 2010
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Contents |
Minipreps using the Qiagen kit
Plasmid extraction
Materials required
- Eppendorf tubes
- Pipettes
- Appropriate overnight cultures
- Buffer P1 (In the fridge)
- Buffer P2
- Buffer N3
- Buffer PB
- Buffer EB
- QIAprep spin column
Procedures
- Overnight culture should have been done the day before. Refer to growing an overnight culture.
- 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 minutes at 13,000 rpm in a table-top microcentrifuge.
- Apply the supernatant to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 30-60 seconds. Discard the flow-through.
- Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 seconds. Discard the flow-through.
- Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60 seconds.
- Discard the flow-through, and centrifuge for an additional 1 minute to remove residual wash buffer.
- 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 minute, and centrifuge for 1 minute.
QIAquick Gel Extraction Microcentrifuge Protocol
Materials
- Scalpel
- Eppendorf tubes
- Pipettes
- QIAquick column
- UV transluminator
- Buffer QG
- Buffer PE
- Buffer EB
- Isopropanol
- 70% ethanol
Protocol
- Before extraction, clean the UV transilluminator and scalpel with 70% ethanol.
- Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. ( Minimise the exposure of the gel to UV as much as possible.)
- Weight the gel slice and add 3 volumes of buffer QG to 1 volume of gel (100 mg ~ 100 µl).
- Incubate at 50°C and invert the tube gently at regular interval until the gel has completely dissolved.
- After the gel has dissolved completely, check that the color of the mixture is yellow.
- Add 1 gel volume of isopropanol to the sample and mix.
- Place a QIAquick spin column in a 2 ml collection tube
- To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min.
- Discard the flow through and place the QIAquick column back into the same tube (max volume: 750 µl).
- Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min. Discard the flow through and place the QIAquick column back into the same tube.
- To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. Place the QIAquick column back into the same tube.
- Centrifuge the column for a further 1 min.
- Transfer the column into a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add 30 µl of Buffer EB to the center of the QIAquick membrane and allow to stand for 1 min.
- Centrifuge the column for 1 min and transfer the eluate to a clean 1.5 ml micriocentrifuge tube.
- To measure the purity of the sample, use a Nanodrop Spectrophotometer.
Go back to our Protocol List