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MACHEREY-NAGEL – 08 / 2013, Rev. 01

Suboptimal

performance

of plasmid

DNA in

enzymatic

reactions

Carry-over of ethanol

•

Make sure that the NucleoSpin

Plasmid EasyPure Column is

completely dry after step 5. Otherwise discard flow-through and

repeat centrifugation.

Elution of plasmid DNA with TE buffer

•

EDTA may inhibit sequencing reactions. Repurify plasmid

DNA and elute with Buffer AE or water. Alternatively, the eluted

plasmid DNA can be precipitated with ethanol and redissolved

in Buffer AE or water.

Not enough DNA used for sequencing reaction

•

Quantify DNA by agarose gel electrophoresis before setting up

sequencing reactions.

Plasmid DNA purification

Содержание NucleoSpin 740727.10

Страница 1: ...Plasmid DNA purification User manual NucleoSpin Plasmid EasyPure August 2013 Rev 01...

Страница 2: ...ww mn net com NucleoSpin Plasmid EasyPure 1 Cultivate and harvest bacterial cells 12 000 x g 30 s 2 Cell lysis 150 L Buffer A1 250 L Buffer A2 RT up to 2 min 350 L Buffer A3 3 Clarification of the lys...

Страница 3: ...ion 6 2 1 Basic principle 6 2 2 Kit specifications 6 2 3 Growth of bacterial cultures 7 2 4 Elution procedures 8 3 Storage conditions and preparation of working solutions 10 4 Safety instructions 11 4...

Страница 4: ...Buffer A2 5 mL 15 mL 75 mL Neutralization Buffer A3 5 mL 20 mL 100 mL Wash Buffer AQ Concentrate 6 mL 6 mL 30 mL Elution Buffer AE 15 mL 15 mL 30 mL Liquid RNase A 2 mg 6 mg 30 mg NucleoSpin Plasmid...

Страница 5: ...e detailed protocol sections of this user manual if the NucleoSpin Plasmid EasyPure kit is used for the first time Experienced users however may refer to the Protocol at a glance instead The Protocol...

Страница 6: ...g step with Buffer AQ Pure plasmid DNA is finally eluted under low ionic strength conditions with slightly alkaline Buffer AE 5 mM Tris HCl pH 8 5 2 2 Kit specifications The NucleoSpin Plasmid EasyPur...

Страница 7: ...roth or CircleGrow can be used In this case bacteria grow faster reach the stationary phase much earlier than in LB medium 12 h and higher cell masses can be reached However this does not necessarily...

Страница 8: ...Recommended culture volumes for NucleoSpin Plasmid EasyPure OD600 1 2 3 4 5 6 Culture volume 15 mL 8 mL 5 mL 4 mL 3 mL 2 mL Note if too much bacterial material is used the lysis and precipitation step...

Страница 9: ...ifuge Apply a second aliquot of elution buffer incubate and centrifuge again Thus about 85 100 of bound nucleic acids are eluted with the standard elution volume at a high concentration Elution Buffer...

Страница 10: ...he preparation Storage of Buffer A2 below 20 C may cause precipitation of SDS If salt precipitate is observed incubate buffer at 30 40 C for several minutes and mix well until all precipitate is redis...

Страница 11: ...skin Ber hrung mit der Haut vermeiden S 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice Bei Ber hrung mit den Augen gr ndlich mit Wasser absp len und Arz...

Страница 12: ...siv sein H 302 Harmful if swallowed Gesundheitssch dlich bei Verschlucken H 315 Causes skin irritation Verursacht Hautreizungen H 317 May cause an allergic skin reaction Kann allergische Hautreaktione...

Страница 13: ...n irritation or a rash occurs Get medical advice attention Bei Hautreizung oder ausschlag rztlichen Rat einholen rztliche Hilfe hinzuziehen P 337 313 Get medical advice attention Bei anhaltender Hautr...

Страница 14: ...A2 Attention Check Buffer A2 for precipitated SDS If a white precipitate is visible warm the buffer for several minutes at 30 40 C until precipitate is dissolved completely Cool buffer down to room t...

Страница 15: ...w through and make sure the spin cup outlet does not touch the wash buffer surface Otherwise repeat the centrifugation step Note To reduce ethanol carry over to a minimum for better performance in dow...

Страница 16: ...oo many bacterial cells used See table 3 for maximum amount of cells Poor plasmid yield Incomplete lysis of bacterial cells See Possible cause and suggestions above No or insufficient amounts of antib...

Страница 17: ...strains used Especially when working with nuclease rich strains keep plasmid preparations on ice or frozen in order to avoid DNA degradation Inappropriate storage of plasmid DNA Quantitate DNA directl...

Страница 18: ...through and repeat centrifugation Elution of plasmid DNA with TE buffer EDTA may inhibit sequencing reactions Repurify plasmid DNA and elute with Buffer AE or water Alternatively the eluted plasmid DN...

Страница 19: ...Buffer A3 740913 1 1 L Buffer AQ Concentrate for 100 mL Buffer AQ 740995 20 mL Buffer AE 740917 1 1 L Liquid RNase A 740397 250 mg Collection Tubes 2 mL 740600 1000 6 3 References Birnboim H C and J D...

Страница 20: ...PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANY CLINICAL USE INCLUDING BUT NOT LIMITED TO DIAGNOSTIC THERAPEUTIC AND OR PROGNOSTIC USE No claim or representations is intended for its use to identi...

Страница 21: ...xpressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are...

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