胶回收 pcr产物回收

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产物纯化简明步骤....................................................................
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 compley 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试剂盒简明步骤（DC3514） （详细内容请参考说明书英文部分） 使用前注意事项：
 使用前请分别在DNA Wash Buffer中加入48 mL(DC3514-01)或者216 mL(DC3514-02) 100%乙醇。
 Buffer GC 在较低温度下易沉淀，若温度较低，出现沉淀，使用前请在37 oC加热几分钟，至沉淀溶解后再使用。
 本试剂盒所有操作步骤均在室温下进行。
 洗脱前，请将Elution Buffer在65oC下预热。
PCR产物纯化离心法操作步骤：
1. 在1倍体积的PCR产物中加入2倍体积的Buffer GC，然后涡旋、混匀。
建议: 小于200 bp的PCR产物，请加入5倍体积的Buffer GC。
2. 转移以上 ≤ 700 μL DNA/Buffer GC 混合溶液至离心柱中，室温下，10,000 xg下离心1分钟，弃废液，将离心柱放回收集管中。（如果DNA溶液/Buffer GC 混合溶液大于700μL，请重复此操作步骤，使剩余的溶液通过离心柱）。
3. 加入500 μL DNA Wash Buffer，室温下在13,000 xg下离心1分钟，倒掉收集管中的废液，将离心柱放回到收集管中。重复步骤3。
注意：确保已将无水乙醇按说明书要求加入到DNA Wash Buffer中。
5. 13,000 x g开盖再次离心1~3分钟，以去除柱中残余的乙醇。
注意：此步操作对DNA的产率多少极为关键。
6. 将柱子放入干净的1.5 mL试管中，向柱中加入在65oC下预热的30-50 μL ElutionBuffer或ddH2O。在室温下放置1分钟。13,000 x g离心1分钟以洗脱DNA。将收集到的洗脱液加入到离心柱中，进行再次洗脱。
建议: 将洗脱缓冲液预热到65oC，加洗脱液后将柱子整个温育5分钟后再离心洗脱可以增加DNA回收效率。 对大于8kb的片段，加洗脱液后将柱子整个温育15-30分钟可以提高回收效率。
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 compley 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.