Modified Qiagen QIAprep Spin Miniprep
Resuspend pelleted bacterial cells in 250μl Buffer P1 and transfer to a micro - centrifuge tube .
Ensure that RNase A has been added to Buffer P1 .
No cell clumps should be visible after resuspension of the pellet .
If LyseBlue reagent has been added to Buffer P1 , vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved .
The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain .
Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times .
Mix gently by inverting the tube .
Do not vortex , as this will result in shearing of genomic DNA .
If necessary , continue inverting the tube until the solution becomes viscous and slightly clear .
Do not allow the lysis reaction to proceed for more than 5 min .
If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition of Buffer P2 .
Mixing should result in a homogeneously colored suspension .
If the suspension contains localized colorless regions or if brownish cell clumps are still visible , continue mixing the solution until a homogeneously colored suspension is achieved .
Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times .
To avoid localized precipitation , mix the solution thoroughly , immediately after addition of Buffer N3 .
Large culture volumes ( e . g .
≥5 ml ) may require inverting up to 10 times .
The solution should become cloudy .
If LyseBlue reagent has been used , the suspension should be mixed until all trace of blue has gone and the suspension is colorless .
A homogeneous colorless sus - pension indicates that the SDS has been effectively precipitated .
Centrifuge for 10 min at 13,000 rpm ( ~ 17,900 x g ) in a table - top microcentrifuge .
A compact white pellet will form .
Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting .
Centrifuge for 30–60 s . Place flow through back into the spin column .
Centrifuge for 30–60 s . Discard the flow - through .
Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s . Discard the flow - through .
This step is necessary to remove trace nuclease activity when using endA + strains such as the JM series , HB101 and its derivatives , or any wild - type strain , which have high levels of nuclease activity or high carbohydrate content .
Host strains such as XL - 1 Blue and DH5®α do not require this additional wash step .
Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s . Discard the flow - through , and centrifuge at full speed for an additional 1 min to remove residual wash buffer .
Important : Residual wash buffer will not be completely removed unless the flow - through is discarded before this additional centrifugation .
Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions .
Place the QIAprep column in a clean 1.5 ml microcentrifuge tube .
To elute DNA , add 20 μl water to the center of each QIAprep spin column , let stand for 1 min , and centrifuge for 1 min .
Elute DNA , add 30 μl water to the center of each QIAprep spin column , let stand for 1 min , and centrifuge for 1 min .
