TIANquick Mini Purification Kit

Transcription

1 TIANquick Mini Purification Kit For purification of PCR products, 100 bp to 20 kb

2 TIANquick Mini Purification Kit (Spin column) Cat no. DP203 Kit Contents Contents Buffer BL Buffer PB Buffer PW Buffer EB DP preps 30 ml 30 ml 15 ml 15 ml Spin Columns CB1 50 Collection Tubes(2 ml) 50 Handbook 1 Storage TIANquick Mini Purification Kit can be stored dry at room temperature (15-25 C)for up to 12 months without showing any reduction in performance and quality. For longer time storage, the kit can be stored at 2-8 C. (Note: Check buffers for precipitate before use and dissolve at 37 C for 10 min if necessary). TIANquick Mini Purification Kit Handbook 1

3 Introduction TIANquick Mini DNA Purification Kit is based on silica-membrane technology. The kit is specially designed for purification of DNA fragments from various reaction solutions (PCR reactions, enzymatic reaction, etc) by removing contaminants of protein, other organic compound, salts and primers, etc. The recovery yield is more than 80% for 100 bp-10 kb DNA fragments. The binding capacity of column CB1 is 5 μg DNA per column. Purified DNA by the kit can be directly used in applications such as restriction enzyme digestion, PCR amplification, sequencing, library screening, ligation and transformation and so on. Important Notes 1. Check BL Buffer before use for salt precipitation. Dissolve any precipitate by warming to 37 C. 2. Buffer BL can improve the absorption capability and stability of the silica membrane. Check Buffers before use for salt precipitation. Redissolve any precipitate by warming at 37 C. After treated with Buffer BL, use the TIANquick Spin Column CB1 as soon as possible (in one day). 3. Increasing the time of absorption and elution could improve recovery efficiency for <100bp and >10kb DNA fragment. 4. The recovery efficiency is related to starting DNA quantity and elution volume. The less starting quantity or elution volume, the less recovery efficiency. 5. The kit has no selectivity for the DNA fragment purify. If you want to purify special DNA fragment selectively and remove some DNA fragment, please use TIANgel Purification Kit. 6. The volume of buffer EB is related with starting DNA quantity TIANquick Mini Purification Kit Handbook 2

4 Starting DNA Quantity Spin Column Buffer EB volume 1-5μg CB μl 5-20μg CB μl 20-30μg CB μl Protocol Add 60ml ethanol (96-100%) to Buffer PW before use (see bottle label for volume). All centrifuge steps are in a conventional tabletop microcentrifuge at room temperature (15-25 C). 1. Column equilibration: add 500ul Buffer BL to the Spin Column CB1. Centrifuge for 1 min at 12,000 rpm (~13,400 g) in a tabletop microcentrifuge. Discard the flow-throw, and then place Spin Column CB1 in the collection tube. 2. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix. It is not necessary to remove mineral oil or kerosene. Note: For example, add 250μl of Buffer PB to 50μl PCR reaction (not including oil). 3. Transfer the mixture to the Spin Column CB1. Let it stand for 2 min at room temperature (15-25 C). Centrifuge for 30~60s at 12,000 rpm (~13,400 g) in a table-top microcentrifuge. Discard the flow-throw, and then place Spin Column CB1 back into the same collection tube. Note: The maximum loading volume of the column is 800 μl. For sample volumes greater than 800 μl simply load again. 4. To wash, add 600 µl Buffer PW to the Spin Column CB1 and centrifuge for 30-60s at 12,000 rpm (~13,400 g). Discard the TIANquick Mini Purification Kit Handbook 3

5 flow-through, and place Spin Column CB1 back in the same collection tube. Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it suggests to let it stand for 2~5min after adding Buffer PW, and then centrifuge. 5. Wash the Spin Column CB1 with 600 µl Buffer PW and centrifuge for 30-60s at 12,000 rpm (~13,400 g). Discard the flow-through, and centrifuge for an additional 2 min to remove residual wash buffer PW. Note: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. 6. Place the Spin Column CB1 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add μl Buffer EB or deionized water (ph ) to the center of membrane, let the column stand for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 g). 7. Alternatively, for increased DNA concentration, add the solution gained from step 6 to the center of membrane again, let the columns stand for 1 min, and then centrifuge. Note: The volume of Buffer EB must be more than 30 ul, or it may affect recovery efficiency. What s more, the ph value of eluted buffer will have some influence in eluting, we suggest chose buffer EB or distilled water (ph ) to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer AE and storing at 20 C is recommended, since DNA stored in water is subject to acid hydrolysis. TIANquick Mini Purification Kit Handbook 4

6 Ordering Information Plasmid DNA Purification Product Size Cat.no. TIANprep Mini Plasmid Kit 50 preps 200 preps DP DP PCR MasterMix Product Size Cat.no. 2 Taq PCR MasterMix (with loading dye) 1 ml 5 1 ml KT KT Taq PCR MasterMix (without loading dye) 1 ml 5 1 ml KT KT Cloning Kit Product Size Cat.no. pgm-t Ligation Kit (contain vector, ligase) 20 µl 60 µl VT VT TIANquick Mini Purification Kit Handbook 5