GRS Plasmid Purification Kit Transfection Grade GK (2 MaxiPreps)

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1 1 GRS Plasmid Purification Kit Transfection Grade GK (2 MaxiPreps) (FOR RESEARCH ONLY) Sample : Expected Yield : Endotoxin: Format : Operation Time : Elution Volume : ml of cultured bacterial cells harbouring target plasmid typically 1000 µg of transfection grade plasmid DNA <0.1 EU/µg DNA gravity flow IEX column within 90 min. 0,5 ml 2,0 ml Product Description The GRS Plasmid Purification Kit Transfection Grade provides an easy and efficient method for the purification of high quality plasmid DNA from cultured bacterial cells. Eluted DNA is free of RNA and endotoxins and is suitable for transfection and other downstream applications including PCR, enzymatic restriction digestion, cloning, in-vivo transcription, and DNA sequencing. Principle The GRS Plasmid Purification Kit Transfection Grade is based on a modified Alkaline Lysis method used to obtain minimal genomic DNA contaminants. RNAse A treatment reduces contamination with RNA. The buffer system is optimized to allow binding of plasmid DNA in the presence of chaotropic salts to the anion-exchange column. Contaminants are removed using a Wash Buffer. The purified plasmid DNA is subsequently eluted using a high salt buffer and then isopropanol-precipitated for desalting. The entire procedure can be completed in within 90 minutes without phenol/chloroform extraction or the need of an ultracentrifuge with a typical DNA yields of 1000µg of transfection grade plasmid.. Quality Control The quality of the GRS Plasmid Purification Kit Transfection Grade is tested on a lot-to-lot basis by isolating plasmid DNA from a 200-ml overnight culture of Escherichia coli DH5α (OD600nm ~2), harbouring pegfp-c2. Yields are typically over 1000 µg of pure plasmid DNA, as determined by spectrophotometer. Caution Some buffers contain harmful irritants. During operation, always wear a lab coat, disposable gloves, and protective goggles. In case of working with potential pathogens, operator has to take appropriate measures according to national and local safety regulations. GRiSP, Lda. does not warrant that cultures containing pathogens are completely inactivated and/or noninfectious by the lysis step. Dispose any waste as potentially infectious sample. Literature Birnboim, HC., and Doly, J. (1979) Nucleic Acids Res. 7,

2 2 Kit Contents Required Components (not included) Resuspension Buffer* 25 ml Isopropanol Lysis Buffer** 25 ml 70% ethanol Neutralization Buffer 25 ml 50-ml centrifuge tubes Column Equilibration Buffer 65 ml TE Buffer or ultrapure water Wash Buffer 35 ml Elution Buffer 25 ml MaxiPrep Columns & Pre-Filter 2 RNAse A (50mg/ml) 50 µl Notes *Add RNAse A to the Resuspension Buffer and mix by inverting 5-10 times. Check the bottle and store at +4ºC for up to 6 months. Storage Store RNAse A and/or Resuspension Buffer containing RNAse A at +4ºC. All other components should be stored at room temperature (15-25ºC). Examine solutions for precipitates before use. Any precipitate (e.g. in the Lysis Buffer) may be re-dissolved by warming the solution to 37ºC followed by cooling to 25ºC.

3 3 Recommended Volumes High-Copy vs Low-Copy Number Plasmids In order to obtain maximum yield and prevent clogging of the filter or the column, it is important to adjust the protocol to the maximum volume of bacterial cell culture that can be used, which depends on the cell density and the type of plasmid. For high-copy number plasmids, the recommended volume is 400ml/OD 600nm. For example, in case of a culture with an OD 600nm of 2,0 the recommended volume corresponds to 400/2,0 = 200ml. For low-copy number plasmids, the recommended volume is 800ml/OD 600nm. For example, in case of a culture with an OD 600nm of 2,5 the recommended volume corresponds to 800/2,5 = 320ml.!!!The protocol for low-copy number plasmids is the same as for high-copy number plasmids (presented on page 4), however, it needs to be taken into consideration that handling higher cell mass requires higher buffer volumes (resuspension, lysis, neutralization). In case OD 600nm x Volume would exceed 400, then one should divide the sample into 2 equal volumes and treat both with Resuspension Buffer, Lysis Buffer and Neutralization Buffer as independent samples. After neutralization, lysates can be pooled and purified by the same column (step 6). This means that if one aims for the maximum yield, processing only cells containing low-copy number plasmids, that this kit does not provide sufficient amounts of resuspension buffer, lysis buffer and neutralization buffer. These buffers can be purchased separately. Alternatively, handling low-copy number plasmids, in case OD 600nm x Volume would not exceed 400, one can follow the protocol without any alteration. Albeit that it is unlikely to achieve the maximum yield, total amount of purified plasmid should be high enough for transfection.

4 4 PROTOCOL FOR PLASMID DNA PURIFICATION 1) Harvest cells by transferring ml* of a culture of bacterial cells harbouring the plasmid of interest to 50-ml or 250-ml centrifuge tubes and centrifuge at 6.000g for 15 minutes (at 4ºC). Discard the supernatant. Use a pipette tip to remove supernatant completely. Repeat harvesting steps for samples >50 ml using the same centrifuge tube or bottle. *) IMPORTANT: before beginning, read first section about recommended volumes on page 2. 2) During centrifugation, remove the MaxiPrep column (with pre-filter already inserted) from the package and place onto a new 50-ml centrifuge tube (not provided). Soak the pre-filter completely, including the rim, with 20ml of Column Equilibration Buffer, and allow the column to completely empty by gravity flow. The pre-filter and the ion-exchange column will be equilibrated simultaneously. Discard the flow-through and place the column back in the 50-ml tube and set aside for step 6. 3) Resuspend the bacterial pellet in 10 ml of Resuspension Buffer (verify that RNAse A has been added), by vortexing, and pipetting up and down, and transfer to a new 50-ml tube (not provided). 4) Add 10 ml of Lysis Buffer (check for precipitates) and mix immediately by gently inverting the tube 5-10 times. DO NOT VORTEX! (vortexing might lead to shearing of genomic DNA which might result in contamination of the final plasmid preparation with genomic DNA fragments). Close the Lysis Buffer bottle immediately to avoid CO 2 acidification. Incubate at room temperature for at least 3 minutes and to ensure homogeneous lysis. Do not exceed 5 minutes. 5) Neutralize the solution by adding 10 ml Neutralization Buffer. Mix immediately by gently inverting the tube 5-10 times. DO NOT VORTEX! Incubate at room temperature for 5 minutes. 6) Following incubation, invert the tube 3 times (to avoid clogging) and apply to the equilibrated pre-filter/maxiprep column. In case of having split the sample (see page 3), pool and apply all. Allow the MaxiPrep column to be emptied completely by gravity-flow. Discard the flow-through, and place the column back onto the 50- ml tube. 7) Wash the MaxiPrep column by adding 10ml of Column Equilibration Buffer to the funnel-shaped rim of the pre-filter. Allow the MaxiPrep column to be emptied completely by gravity-flow.

5 5 8) Discard the flow-through, and place the column back onto the 50-ml tube. Also discard now the pre-filter. 9) Wash the MaxiPrep column by adding 15ml of Wash Buffer. Allow the column to empty completely by gravity flow. Discard the flow-through. and place the column back into a new 50-ml centrifuge tube. 10) Elute plasmid DNA by adding 10ml of Elution Buffer and allow the column to empty completely by gravity flow. Discard the column once it has been completely emptied. 11) Precipitate plasmid DNA by adding 7,5ml (0,75 volumes) of isopropanol (2- propanol). Mix the tube by inverting times, and allow to stand for 2 minutes at room-temperature. Centrifuge at g for at least 15 min (preferably 30 min) at +4ºC. 12) Carefully remove the supernatant and then wash the pellet with 5ml of 70% ethanol. Centrifuge at g for at least 5 min (preferably 10 min) at +4ºC. 13) Carefully remove the supernatant and then air-dry the pellet for 10 minutes. Once the pellet is dry, resuspend the DNA pellet in the desired volume (e.g. 0,5-2,0 ml) of TE (or ddh 2 O). Place the tube for 5-10 minutes in a 60ºC water bath to dissolve the DNA completely.

6 6 TROUBLESHOOTING 1. Low Yield Buffers and Cells i. If precipitates have formed in the Lysis Buffer, warm in a 37ºC water bath, followed by gentle shaking to dissolve all precipitates and allow to cool down to room temperature. ii. Make sure RNAse A is added to the Resuspension Buffer and that the bottle is stored at 2-8ºC for up to 6 months. iii. Inoculate ml of LB medium with only a single freshly isolated E.coli colony of cells harbouring the plasmid of interest. All media, solid and liquid, should contain the appropriate antibiotic(s). Do not use overgrown bacterial cultures (max 16 hours at 37ºC with rpm shaking). Check plasmid content in cleared lysate. Incomplete Lysis of bacterial cells i. During harvesting, make supernatant is completely removed using a pipet tip. ii. Resuspend the cell pellet completely by vortex or by pipetting. iii. An OD600 nm of 2 to 6 units is highly recommended. iv. Too much cells were used: In case of OD600 nm x volume exceeds 400, split samples as described on page 3. Incomplete DNA rehydration i. If using water to dissolve the DNA pellet, make sure that the ph is above 7,5. Water should be prepared freshly as CO 2 from the environment could cause acidification and lower the DNA s solubility. 2. Low Quality Low performance in downstream applications i. Residual ethanol contamination interferes with downstream applications. Following the precipitation step with isopropanol and the wash step with 75% ethanol, ensure that all ethanol has been removed by pipetting and air drying. ii. In case of genomic DNA contamination (which can be detected by gel analysis), make sure that mixing steps are done gently to prevent smearing. Also do not use overgrown bacterial cultures. iii. RNA contamination might be due to incorrect storage of the Resuspension Buffer, or RNAse A was not added. Add new RNAse A to the Resuspension Buffer. GRiSP Research Solutions Rua Alfredo Allen, UPTEC Edifício Central Porto, PORTUGAL Tel: Fax:

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