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Molecular Biology Kits

PCR Taq

1. What are the functions of Taq DNA polymerase?
2. What characteristics does GenScript Taq DNA polymerase have?
3. What is the recommended enzyme amount when using Taq DNA Polymerase or Green Taq DNA Polymerase?
4. How is Taq different from Green Taq?
5. Can Taq DNA Polymerase or Green Taq be used to amplify GC-rich amplicons?
6. Can Taq DNA Polymerase be used in other buffers?
7. Which buffer should I use if I want to control the level of magnesium (Mg2+) in the reaction? Does the presence of Mg2+ inhibit PCR?
8. How should I set up an amplification reaction using Taq DNA Polymerase?
9. What is the proper concentration for a routine PCR reaction?
10. What is the maximum product length that can be made by GenScript's Taq DNA Polymerase?
11. What type of DNA end results from a primer extension reaction or a PCR using Taq DNA Polymerase?
12. Why is there no product when visualized on an agarose gel?
13. The product sequence doesn't completely match the expected sequence. How can this result be improved?
14. When should Taq DNA Polymerase be used in a primer extension reaction or for PCR?
15. Will the 5'→3' flap endonuclease activity of Taq DNA Polymerase degrade primers?
16. Where can I find help troubleshooting my PCR?

PCR Cloning Kit

1. What are the differences between the GenBuilderkit and the PCR Cloning Kit?
2. Technical information is not available in the manual, why is that?
3. Why don't I generate sufficient colonies when the GenBuilder™ reaction is transformed?
4. Why do I generate incorrect colonies when the GenBuilder™ reaction is transformed?
5. What factors need to be considered when designing the primer?
6. What should the purity of my primer be to be compatible with the GenBuilder™ cloning method?
7. Can multiple fragments be cloned into a single vector using this kit?
8. Can I use GenBuilder™ PCR Cloning Kit with my own vectors?
9. What factors need to be considered when using GenBuilder™ PCR Cloning Kit with customers' vector?
10. Will the GenBuilder™ reaction work more efficiently if I use primers that contain a longer than 15 base region of homology?
11. What is the stability of the enzyme included in GenBuilder™ PCR Cloning Kit?

Plasmid Purification

1. Why is my plasmid DNA yield low?
2. Can the QuickClean II plasmid Miniprep Kit be used for isolating plasmid DNA from mammalian cells?
3. What is the recommended culture medium for the Plasmid miniprep kit?
4. How do I know if my plasmid is a high- or low copy number type?
5. How to resolve genomic DNA contamination in my plasmid prep?
6. Can I use QuickClean Miniprep kits for low-copy plasmids and cosmids?
7. Are GenScript buffers with identical names in different kits the same?
8. Will QuickClean II PCR Purification Kit remove fluorescent dyes from real-time PCR reactions?
9. Is it necessary to repeat the wash procedure?
10. Can I use TE buffer or water to elute DNA from the column?
11. What additional consumables does the user need?
12. Can I extract and purify DNA from gels using TAE running buffer?
13. Can I extract and purify DNA from low melting point (LMP) Agarose gels?

PCR Taq

Answers:

Yes,QuickCleanIIPCRPurificationKitsremoveSYBRGreendyeefficientlyfromthereal-timePCRreaction.ThekitwillalsoremovefluorescentdyelabeleddNTPusedforPCRDNAlabeling,suchasCy3-dUTP,etc.

Checkifbacteriacellswereresuspendedcompletely.

HowtoresolvegenomicDNAcontaminationinmyplasmidprep?

Yes.QuickCleanIIPCRorGelExtractionKitcanbeusedtoextractandpurifyDNAfromlowmeltingpoint(LMP)gels.

Isitnecessarytorepeatthewashprocedure?

WhyismyplasmidDNAyieldlow?

IncubatetheElutionBufferat30-60°C，thisshouldhelpincreaseyields.

AreGenScriptbufferswithidenticalnamesindifferentkitsthesame?

IfthereisRNAintheresult,addmoreRNaseAtoResuspensionBuffertoafinalconcentration100μg/ml.

Yes,GenScriptbuffersthathaveexactlythesamename,arechemicallyidenticalandcanbeexchangedbetweenkits.Forexample,washsolutionandelutionbufferfromtheQuickCleanIIPCRPurificationKitareexactlythesameaswashsolutionandelutionbufferfromQuickCleanIIPCRGelExtractionKit.

CanIextractandpurifyDNAfromgelsusingTAErunningbuffer?

Whatadditionalconsumablesdoestheuserneed?

CanIuseTEbufferorwatertoeluteDNAfromthecolumn?

ElutionBufferis2.5mMTris-HClpH8.5.TEbufferorwatercanalsobeused,butyieldwillbeslightlylower.ThepHoftheelutionsolutioniscritical;bufferswithahigherpHsuchas8.5orabovewillbemoreefficienttoeluteDNAfromthecolumn.

Pleasecheckifthebacteriawasculturedproperly.

Ethanolisneededtopreparethewashsolutionforallthreekits.IsopropanolisalsoneededforQuickCleanIIPCRorGelExtractionKit.

CanIextractandpurifyDNAfromlowmeltingpoint(LMP)Agarosegels?

InthecaseofalowratioofOD260-320/OD280-320,theremaybeproteincontamination.InthiscasepleaseaddNeutralizationBuffer,andthencentrifugebufferwithsufficientrotatingspeed,thustomakeprecipitationcompact;becarefultopipettesupernatantandavoidpipettingtheprecipitate.

ObtainnoplasmidDNA

TheDNAelutedfromthecolumnispureenoughformostdownstreamapplications.However,ifthedownstreamapplicationsaresensitivetosaltcarryover,itisbettertowashthecolumntwicepriortoelution.

Absorbanceisthedifferenceofthesamplefromtheblank,pleaseusetheElutionBuffertoadjusttheblanktoazerovalueanduseittodilutethesample.

Problem

Solution

No.TheKitprovidesafast,simple,andcost-effectiveplasmidminiprepmethodfor1–5mlofovernightculturesofE.Coli.

HowdoIknowifmyplasmidisahigh-orlowcopynumbertype?

IftheratioofOD260/OD230istoolow,washthespincolumnforonemoretime.

IfthereisnoplasmidDNAintheelutionbuffer,pleasecheckwhethertheethanolhadbeenaddedtowashbufferaccordingtothevolumemarkedonbottlelabel.

Electrophoresisproblem

Checkifthebacteriawasculturedproperly.

LBculturemedium

WhatistherecommendedculturemediumforthePlasmidminiprepkit?

Absorbanceproblem

IfthereisgenomicDNAintheresult,invertthetubegently(step3and4).

CantheQuickCleanIIplasmidMiniprepKitbeusedforisolatingplasmidDNAfrommammaliancells?

CanIuseQuickCleanMiniprepkitsforlow-copyplasmidsandcosmids?

Yes.QuickCleanIIPCRorGelExtractionKitcanbeusedtoextractandpurifyDNAfromgelsusingeitherTBEorTAEastheelectrophoresisbuffer.

Forexample,aseriesofPUCvectorishigh-copy-numberplasmid.Youcanknowthedifferenttypeofplasmidfromtheirname.

IftheratioofOD260-320/OD280-320istoohigh,addmoreRNaseAtoResuspensionBuffertoafinalconcentration100μg/ml.

IfgettinggenomicDNAcontaminationintheprep,invertthetubegentlyafteraddingtheLysisBuffer.

WillQuickCleanIIPCRPurificationKitremovefluorescentdyesfromreal-timePCRreactions?

Pleasecheckifthebacteriacellswereresuspendedcompletely.3.IncubatetheElutionBufferat30～60°C，tohelpincreasetheyields.

Thekitcanpurifytheplasmidfromlow-copyplasmids,butfromcosmids.

LowplasmidDNAyields

Plasmid Purification

Answers:

1. Why is my plasmid DNA yield low?

1. Check if the bacteria was cultured properly.
2. Check if bacteria cells were resuspended completely.
3. Incubate the Elution Buffer at 30-60°C，this should help increase yields.

2. Can the QuickClean II plasmid Miniprep Kit be used for isolating plasmid DNA from mammalian cells?

No. The Kit provides a fast, simple, and cost-effective plasmid miniprep method for 1–5ml of overnight cultures of E. Coli.

3. What is the recommended culture medium for the Plasmid miniprep kit?

LB culture medium

4. How do I know if my plasmid is a high- or low copy number type?

For example, a series of PUC vector is high-copy-number plasmid. You can know the different type of plasmid from their name.

5. How to resolve genomic DNA contamination in my plasmid prep?

If getting genomic DNA contamination in the prep, invert the tube gently after adding the Lysis Buffer.

6. Can I use QuickClean Miniprep kits for low-copy plasmids and cosmids?

The kit can purify the plasmid from low-copy plasmids, but from cosmids.

7. Are GenScript buffers with identical names in different kits the same?

Yes, GenScript buffers that have exactly the same name, are chemically identical and can be exchanged between kits. For example, wash solution and elution buffer from the QuickClean II PCR Purification Kit are exactly the same as wash solution and elution buffer from QuickClean II PCR Gel Extraction Kit.

8. Will QuickClean II PCR Purification Kit remove fluorescent dyes from real-time PCR reactions?

Yes, QuickClean II PCR Purification Kits remove SYBR Green dye efficiently from the real-time PCR reaction. The kit will also remove fluorescent dye labeled dNTP used for PCR DNA labeling, such as Cy3-dUTP, etc.

9. Is it necessary to repeat the wash procedure?

The DNA eluted from the column is pure enough for most downstream applications. However, if the downstream applications are sensitive to salt carryover, it is better to wash the column twice prior to elution.

10. Can I use TE buffer or water to elute DNA from the column?

Elution Buffer is 2.5 mM Tris-HCl pH 8.5. TE buffer or water can also be used, but yield will be slightly lower. The pH of the elution solution is critical; buffers with a higher pH such as 8.5 or above will be more efficient to elute DNA from the column.

11. What additional consumables does the user need?

Ethanol is needed to prepare the wash solution for all three kits. Isopropanol is also needed for QuickClean II PCR or Gel Extraction Kit.

12. Can I extract and purify DNA from gels using TAE running buffer?

Yes. QuickClean II PCR or Gel Extraction Kit can be used to extract and purify DNA from gels using either TBE or TAE as the electrophoresis buffer.

13. Can I extract and purify DNA from low melting point (LMP) Agarose gels?

Yes. QuickClean II PCR or Gel Extraction Kit can be used to extract and purify DNA from low melting point (LMP) gels.

Problem	Solution
Obtain no plasmid DNA	If there is no plasmid DNA in the elution buffer, please check whether the ethanol had been added to wash buffer according to the volume marked on bottle label.
Low plasmid DNA yields	Please check if the bacteria was cultured properly. Please check if the bacteria cells were resuspended completely. 3. Incubate the Elution Buffer at 30～60°C，to help increase the yields.
Absorbance problem	Absorbance is the difference of the sample from the blank, please use the Elution Buffer to adjust the blank to a zero value and use it to dilute the sample. If the ratio of OD260/ OD230 is too low, wash the spin column for one more time. In the case of a low ratio of OD260-320/ OD280-320, there may be protein contamination. In this case please add Neutralization Buffer, and then centrifuge buffer with sufficient rotating speed, thus to make precipitation compact; be careful to pipette supernatant and avoid pipetting the precipitate. If the ratio of OD260-320/ OD280-320 is too high, add more RNase A to Resuspension Buffer to a final concentration 100 μg/ml.
Electrophoresis problem	If there is genomic DNA in the result, invert the tube gently (step 3 and 4). If there is RNA in the result, add more RNase A to Resuspension Buffer to a final concentration 100 μg/ml.