膠回收 pcr產物回收
Table of Contents
Contents……………………………………………………………...
1
Introduction.........................................................................................
2
Storage andStability.....................................................…..................
2
KitContents........................................................................................
2
SafetyInformation..............................................................................
2
BeforeStarting....................................................................................
3
PCR Purification SpinProtocol..........................................................
4
PCR Purification Vacuum/SpinProtocol...................................…….
5
PCR產物純化簡明步驟....................................................................
6
Trouble ShootingGuide..................................................................…
7
Limited use andwarranty....................................................................
8
Page 2 Biomiga EZgeneTM Cycle Pure kit
Introduction This fast and reliable kit is designed to purify DNAfragments from PCR, RFLP, phosphorylation, labeling, ligation,hybridization and other enzymatic reactions. DNA fragments from 70bp to 20 kb can be purified using the ezBindTM mini column withover 80-90 % recovery. Storage and Stability All components can bestored at room temperature. All kit components are guaranteed for12 months from the date of purchasing. Kit Contents
Catalog#
DC3514-00
DC3514-01
DC3514-02
Preps
4
50
250
Buffer GC
2.5 mL
28 mL
125 mL
DNA Wash Buffer*
2 mL
12 mL
54 mL
Elution Buffer
1 mL
10 mL
20 mL
ezBind Columns
4
50
250
User Menu
1
1
1
*Add 48 mL or 216 mL 100% ethanol to DNA Wash Buffer before use.Safety Information Buffer GC contains acidic acid and chaotropicsalts, which may form reactive compounds when combines with bleach.Do not add bleach or acidic solutions directly to the preparationwaste.
Biomiga EZgene Page 3 TM Cycle Pure kit
Before Starting Prepare all components and get all necessarymaterials ready by examining this instruction booklet and becomefamiliar with each steps. Important
? Add 48 mL (DC3514-01) 100% ethanol to DNA Wash Buffer beforeuse.
? Add 216 mL (DC3514-02) 100% ethanol to DNA Wash Buffer beforeuse.
? Pre-warm aliquots of Elution Bufer or ddH2O at 55-60°Cwaterbath.
Materials supplied by users
? Tabletop microcentrifuge and 1.5 mL microtubes.
? Vacuum manifold if vacuum protocol is applied.
? 96~100 % ethanol.
Perform all steps including centrifugation at room temperature!
Page 4 Biomiga EZgeneTM Cycle Pure kit
PCR Purification Spin Protocol
1. Add 2 volumes of Buffer GC to 1 volume of the PCR reaction andmix completely by vortexing. Briefly spin the tube to collect anydrops from the inside wall and tube lid.
Note: For PCR products < 200 bp in length, add 5 volumes ofBuffer GC to 1 volume of PCR reaction.
2. Transfer up to 700 μL DNA/Buffer GC mixture to a spin columnwith a collection tube. Centrifuge at 13,000 x g for 1 min at roomtemperature. Discard the flow-through and put the column back tothe collection tube. Repeat this step to process the remainingsolution.
3. Add 500 μL DNA Wash Buffer to the column and centrifuge at13,000 x g for 1 min at room temperature. Discard the flow throughand insert the column, with the lid open, back to the collectiontube.
Note: Ensure that ethanol has been added to DNA Wash Buffer asinstructed before use.
4. Repeat step 3. Centrifuge the empty DNA column, with the lidopen, at top speed for 1-3 min to remove the residual ethanol inthe column.
Note: The residual ethanol will be removed more efficiently withthe column lid open during centrifugation.
5. Place the column into a clean 1.5 mL micocentrifuge tube and add30-50 μL Elution Buffer or ddH2O to the center of the column.Incubate at room temperature for 1 min. Centrifuge at 13,000 x gfor 1 min to elute the DNA.
Note: Pre-warm elution buffer or ddH20 at 65°C and incubate thecolumn at 65°C for 5 min after adding elution buffer or ddH20 willincrease the DNA yield. Note: The first elution normally yields60-70% of the DNA bound. Reload the eluted DNA solution to thecolumn for a second elution will yield another 20% of the DNA thatmakes the total yield up to 90%.
Biomiga EZgene Page 5 TM Cycle Pure kit
PCR Purification Vacuum/Spin Protocol
1. Follow the instruction described on step 1 on page 4. Brieflyspin the tube to collect any drops from the inside wall and tubelid. For PCR products less than 200 bp, add 5 volumes of BufferGC.
2. Prepare the vacuum manifold according to manufacturer’sinstructions. Attach the spin column to the manifold.
3. Load the PCR reaction / Buffer GC solution to a spin column thatis attached to the manifold. Turn on the vacuum to let the solutionpass through the column.
4. Wash the column by adding 500 μL DNA Wash Buffer.
5. Repeat step 4. Vacuum the column for 1 min.
6. Put the column, with the lid open, in a collection tube, andspin at top speed for 2 min.
Note: The residual ethanol will be removed more efficiently withthe column lid open during centrifugation.
7. Put the column to a clean 1.5 mL tube and add 30–50 μL ElutionBuffer or ddH2O to the column. Incubate at room temperature for 1min. Centrifuge the tube at 13,000 x g to elute DNA.
Note: Pre-warm elution buffer or ddH20 at 65°C and incubate thecolumn at 65°C for 5 min after adding elution buffer or ddH20 willincrease the DNA yield.
Note: The first elution normally yields 60-70% of the DNA bound.Reload the eluted DNA solution to the column for a second elutionwill yield another 20% of the DNA that makes the total yield up to90%.
Page 6
Biomiga EZgeneTM Cycle Pure Kit
PCR試劑盒簡明步驟（DC3514） （詳細內容請參考說明書英文部分） 使用前注意事項：
? 使用前請分別在DNA Wash Buffer中加入48 mL(DC3514-01)或者216 mL(DC3514-02) 100%乙醇。
? Buffer GC 在較低溫度下易沉淀，若溫度較低，出現沉淀，使用前請在37 oC加熱幾分鐘，至沉淀溶解后再使用。
? 本試劑盒所有操作步驟均在室溫下進行。
? 洗脫前，請將Elution Buffer在65oC下預熱。
PCR產物純化離心法操作步驟：
1. 在1倍體積的PCR產物中加入2倍體積的Buffer GC，然后渦旋、混勻。
建議: 小于200 bp的PCR產物，請加入5倍體積的Buffer GC。
2. 轉移以上 ≤ 700 μL DNA/Buffer GC 混合溶液至離心柱中，室溫下，10,000 xg下離心1分鐘，棄廢液，將離心柱放回收集管中。（如果DNA溶液/Buffer GC 混合溶液大于700μL，請重復此操作步驟，使剩余的溶液通過離心柱）。
3. 加入500 μL DNA Wash Buffer，室溫下在13,000 xg下離心1分鐘，倒掉收集管中的廢液，將離心柱放回到收集管中。重復步驟3。
注意：確保已將無水乙醇按說明書要求加入到DNA Wash Buffer中。
5. 13,000 x g開蓋再次離心1~3分鐘，以去除柱中殘余的乙醇。
注意：此步操作對DNA的產率多少極為關鍵。
6. 將柱子放入干凈的1.5 mL試管中，向柱中加入在65oC下預熱的30-50 μL ElutionBuffer或ddH2O。在室溫下放置1分鐘。13,000 x g離心1分鐘以洗脫DNA。將收集到的洗脫液加入到離心柱中，進行再次洗脫。
建議: 將洗脫緩沖液預熱到65oC，加洗脫液后將柱子整個溫育5分鐘后再離心洗脫可以增加DNA回收效率。 對大于8kb的片段，加洗脫液后將柱子整個溫育15-30分鐘可以提高回收效率。
Page 7
Biomiga EZgeneTM Cycle Pure Kit
Trouble Shooting Guides
Problems
Possible reasons
Suggested improvements
Low DNA yield
1. Not enough Buffer GC
2. Reused electrophoresis buffer with increased pH.
Determine the volume of Buffer GC to be used correctly asinstructed. For PCR products less than 200 bp, add 5 volumes BufferGC. Use fresh electrophoresis buffer.
No DNA yield
Forgot to add ethanol to DNA Wash Buffer
Add absolute ethanol to DNA Wash Buffer as instructed beforeuse.
DNA sample floats out of well while loading agarose gel
Ethanol was not completely removed from the column following washstep
After the wash step, centrifuge the empty column with the lid openat top speed for 1-3 min. Repeat once.
Page 8
Biomiga EZgeneTM Cycle Pure Kit
Limited Use and Warranty This product is warranted to perform asdescribed in its labeling and in Biomiga’s literature when used inaccordance with instructions. No other warranties of any kind,express or implied, including, without limitation, impliedwarranties of merchantability or fitness for a particular purpose,are provided by Biomiga. Biomiga’s sole obligation and purchaser’sexclusive remedy for breach of this warranty shall be, at theoption of Biomiga, to replace the products, Biomiga shall have noliability for any direct, indirect, consequential, or incidentaldamage arising out of the use, the results of use, or the inabilityto use it product.
For technical support or learn more product information, pleasecontact us at (858) 603-3219 or visit our website at www.biomiga.com * FOR RESEARCH USEONLY.