Latest revision as of 12:54, 2 August 2010

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Minipreps using the Qiagen kit

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.

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 {{Team:Newcastle/mainbanner}}
-==Minipreps==
+=Minipreps using the Qiagen kit=
-===Gel 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
-#Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
+==Procedures==
-#Weigh the gel slice in the colourless tube. Add 3 volumes Buffer QG to 1 volume of gel slice in a colourless tube (100mg~10µl)
+# Overnight culture should have been done the day before. Refer to [[Team:Newcastle/Growing an overnight cultures| growing an overnight culture]].
-#Incubate at 50°C for 10 min (or until the gel slice has completely dissolved)vortex every 2-3minutes
+# Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
-#After the gel slice has dissolved completely, check the colour of the mixture is yellow
+# Add 250 μl Buffer P2 and mix throughly by inverting the tube 4-6 times.
-#Add 1gell volume of isopropanol to the sample and mix
+# Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
-#Place QG quick spin column in 2ml collection tube
+# Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge.
-#Add sample and spin for 1 minute (discard flow through)
+# Apply the supernatant to the QIAprep spin column by decanting or pipetting.
-#Add QG buffer and spin for 1min (discard flow through)
+# Centrifuge for 30-60 seconds. Discard the flow-through.
-#Add PE buffer and spin for 1min (discard flow through)(13000rpm)
+# Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 seconds. Discard the flow-through.
-#Place spin column in clean tube(1.5ml)
+# Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60 seconds.
-#Add elution buffer (50µl)spin for 1 min
+# Discard the flow-through, and centrifuge for an additional 1 minute to remove residual wash buffer.
-#If using a gel to analyse add loading dye and mix (pipet up and down)
+# 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.
+'''Go back to our [[Team:Newcastle/Protocol list|Protocol List]]'''
+{{Team:Newcastle/footer}}

Team:Newcastle/Minipreps

From 2010.igem.org

Latest revision as of 12:54, 2 August 2010

Minipreps using the Qiagen kit

Materials required

Procedures