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Genomic DNA isolation kit with Tini spin column, 50 preps

 Purification of Genomic DNA from Small Animal Tissue/Tail samples 

(Cat#601) 

Kit Contents and Storage Conditions: Components 

601 (50 preps) 

Storage 

ACL Solution 

PBS Solution 

AB solution* 

WASH Solution 

Elution Buffer 

ProteinaseK (20mg, lyophilized powder) 

DNA Tini Spin Column/ with collection tubes 

20ml 

20ml 

20ml 

12ml 

5ml 

20mg 

50 

RT (room temperature) 

RT 

RT 

RT 

RT 

-20oC 

RT 

 *Tini Spin columns can be ordered separately for leftover solutions (cat#EZC106, $39 for 50 columns) 

Safety Information: 

AB Solution contains guanidine hydrochloride: harmful, irritant. Risk and safety phrases:* R22-36/38, S13-26-36-46 

proteinase K: sensitizer, irritant. Risk and safety phrases:* R36/37/38-42/43, S23-24-26-36/37 

Description: 

This kit is designed for fast isolation of genomic DNA from small amount of animal tissue (including mouse tails) without organic solvent extraction or ethanol precipitation. The typical size is ranged from 100bp-50kb. The kit contains a membrane embedded DNA Tini spin column for binding up to 20g of DNA and for removing salts, enzymes, proteins, and all other impurities. The recommended sample size <5x105 cell and <5mg tissue (for genotyping with mouse tail: <0.2 cm, rat tail: <0.1cm). This kit can also be used for concentration of genomic DNA since the elution volume of Tini Spin Column can as low as 5l. For larger sample use kit EZC205 with Mini Spin Columns or buy bulk DNA Mini Spin Column (EZC101, $36 for 100 columns) 

Applications: 

1. Genomic DNA preparation from different sources: 

(a) Blood (b) Various animal tissues (c) Mouse and Rat tails. (D) Saliva (E) water sample 

2. Highly purified genomic DNA is ready to use in the following applications: 

(a)PCR (b) Southern blot (c) Analysis by pulsed-field electrophoresis (d) Restriction enzymes digestion 

Features: 

√ Fast and High yield √ No phenol / chloroform extractions √ No ethanol precipitation 

√ Yields fully hydrated genomic DNA √ Columns are sold separately for leftover solutions 

Procedures for isolation of Genomic DNA from small amount of Animal Tissues/Tails 

This protocol is designed for isolation of genomic DNA from small amount of animal tissues and mouse tails using DNA Tini Spin Column. Usually mouse tails have insignificant amount of RNA, so RNase A digestion can be omitted. Transcriptional active tissues such as liver and kidney contain high levels of RNA, RNase A may be used to digest RNA (before adding BD buffer). RNA does not affect PCR. 

 Important Notes: 

In order to obtain optimal genomic DNA yield and purity, it is essential to use the correct amount of starting material. Here is the recommendation for maximum amounts of starting material:
Sample 

Amount for Mini Spin Column 

Amount for Mini Spin Column 

Animal tissue 

Mammalian Blood 

Bird or fish blood (with nucleated erythrocytes) 

Mouse Tails (for genotyping) 

Rat Tails (for genotyping) 

Cultured Cells 

Bactuerial 

25mg 

100l 

10l 

0.3cm 

0.3cm 

5x106 

2x109 

10mg 

0.1cm 

0.1cm 

5x105 

Animal tissue size and estimated weight: for most animal tissues Tissue size (LxW) (mm) 

Thickness (mm) 

Estimated weight (mg) 

1.5mmx1.5mm 

1.7mmx1.7mm 

2.0mmx2.0mm 

2.5mmx2.5mm 

3.0mmx3.0mm 

3.5mmx3.5mm 

1-2mm 

1-2mm 

1-2mm 

1-2mm 

1-2mm 

1-2mm 

1mg 

3mg 

5mg 

7mg 

10mg 

15mg 

Things to do before starting: 

1. Buffer ALC may form precipitates upon storage. Warm to 55oC if necessary. 

2. Add 1ml sterilized water to proteinase K to make stock solution at 20mg/ml (keep at -80oC for long term storage). 

3. Add 48ml of 100% ethanol to 12ml of WASH buffer. 

4. If using frozen tissue, equilibrate the sample to room temperature. Avoid freeze/thaw cycle of samples since this will lead to reduced DNA size. 

5. Prepare ALC+proteinase K mixture: add 100l of proteinase K stock into1.5ml ALC. This is good for 5 DNA preps for tails. 

Procedures: 

1. Cut up to 0.1cm mouse tails and place in a 1.5ml microcentrifuge tube, For tissue samples, cut up to 10mg tissue (up to 5mg spleen) into small pieces and place into tube. 

2. Add 140l ALC buffer+proteinase K and incubate the tube at 550C till tissue or tail is completely lysed (2h to overnight). Ensure the tissue is completely immersed in the buffer mix. (Option to treat the sample with RNase A (not included): 2ul of 100mg/ml to each tube and incubate for 2 min at room temperature). 

3. When lysis completes, cool to room temperature, votex for 20 seconds and centrifuge at full speed for 2 min. 

4. Pipette 120l of supernatant into Tini spin Column and add equal volume (120l) of AB solution. Mix by inverting the column 4-6 times, and then centrifuge in a microcentrifuge for 1 min at full speed. Discard flow-through. 

5. Add 125ul of WASH buffer (ethanol added) to the column and centrifuge at full speed for 1 minute. Discard flow-through and repeat this step one more time. 

6. Centrifuge the tube for additional minute to remove any residual wash solution (This step is important). 

7. Place the column into a clean 1.5ml centrifuge tube. 

8. Add 10-50l ddH2O (preheated at 650C) to the center of the column and incubate at room temperature for 5 min. Centrifuge at 10,000xg (~13,000rpm) for 1 minute to elute the genomic DNA. Additional elution step may need for better recovery of genomic DNA. 

Procedures for isolation of Genomic DNA from Small Amount of Animal/Human Cell and Blood. 

This protocol is design for isolation of genomic DNA from small amount of animal/human cell and blood lysate using DNA Tini spin column (Cat#EZC106) 

Cell numbers in Different Sizes of Multiwall Culture Plates and Dishes Multiwell plates 

Number of Cells 

Culture Dishes 

Number of Cells 

96-well 

48-well 

24-well 

12-well 

6-well 

4-5x104 

1x105 

2.5x105 

5x105 

>1x106 

35mm dish 

60mm dish 

100mm dish 

145-150mm 

40-50ml Flask 

250-300 ml Flask 

650-750ml Flask 

1x106 

2.5x106 

7x106 to 1x107 

2x107 

3x106 

1x107 

2x107 

1. For adherent cells (up to 5x105), remove the medium and harvest cells by trypisinization or method of your choice. Wash the cells with 250l PBS solution. Resuspend cells in 150l ACL buffer+ proteinase K. 

2. For suspension cells (up to 5x105), harvest the cell by centrifugation. Resuspend cells in 150l ACL buffer+proteinase K. 

3. For fresh or frozen anticoagulated mammal blood sample (nonnucleated erythrocytes), add 135l blood sample to a sterile microcentrifuge tube, add 7.5l of 10% SDS (final 0.5%), mix thoroughly by votexing or pipetting to yield homogeneous solution. If using <135l blood sample, adjust the sample volume to135l using PBS and then add SDS. 

4. For fresh or frozen nucleated erythrocytes (blood from birds, fish or frogs), add 5l anticoagulated blood to ~150l ALC buffer+proteinase K, and mix thoroughly by votexing or pipetting to yield homogeneous solution. 

5. Option to treat the sample with RNase A: add 2l of 100mg/ml to each tube and incubate for 2 min at room temperature. 

6. Incubate at 550C for 10-20min to promote protein digestion. 

7. Follow step 3-8 from the protocol above. 

PRODUCTS ARE INTENDED FOR BASIC SCIENTIFIC RESEARCH ONLY! 

NOT INTENDED FOR HUMAN OR ANIMAL USE! 


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