GenotypingTransgenicRodentsbyPCR

ThisishowwetestmiceandratsforthepresenceofthetransgenebyPCR.ItisprovidedforthoseinvestigatorswhoareunfamiliarwithDNAgenotypingandwhoneedtoestablishagenotypingmethod.

1.QuantitatetailDNA

2.Prepare20µlaliquotsoftailDNAdilutedto200ng/microliterinautoclavedwater.

3.HeatDNAfor1minuteinadrybathsetfor95degreesC.spindowntubesplaceonice

4.PreparePCRcocktail

5.Aliquot24.0microliterofcocktailintoPCRtubes.

6.Add1.0microliterofheattreatedDNAtoPCRtube.(iftailDNAislessthan200ng/microliterthenadd200ngofDNAinavolumeupto3microliters)

7.FinalvolumeofPCRreactionis25.0microliters.

8.Overlaywithmineraloilandplaceintothermalcycler.

9.Amplify30to35cycles.

10.Analyzeonappropriateethidiumbromidecontainingagarosegel.weanalyzeupto60samplesona20X20cm1.5%agarosegelinTBE

Tips:Prepareacocktailwithanextra10%toaccountforpipettingerrors.RemembertoincludeanegativecontrolofwateronlyandapositivecontrolofknowntransgenicmouseDNAorlinearizedplasmidDNAdilutedinmousetailDNAtotheonecopylevel.RemembertoincludeaninternalpositivecontroltoensurethattheDNAsampleis"amplifiable."Forthispurposeweuseprimersformousebetaglobin.Primersthatamplifyanysinglecopymousegenemayalsobeused.Forexample,amplificationofbetaglobinindicatesthattheDNAiscleanand"amplifiable."Thiswillpreventfalsenegativeresultsandpositiveanimalswillnotbeaccidentallydiscarded.Internalpositivecontrolprimersmaybecombinedwithtransgenespecificprimersifthetwoprimersetsdonotinterferewitheachother.Useprimerpickingsoftware.WehavehadverygoodresultswithPrimer3.

PCR方法相关产品:

• 电泳设备
• 紫外设备
• 普通PCR仪
• 定量PCR仪
• PCR/RT-PCR/qPCR试剂
• PCR引物
• PCR试剂
• PCR对照
• 特异性PCR试剂盒
• PCR克隆试剂盒
• RNA
• RNase检测/去除
• RT-PCR试剂
• RT-PCR标准品
• 定量PCR试剂
• 定量PCR标记
• 总RNA分离纯化盒
• PCR产物纯化
• 核酸酶
• 聚合酶
• 反转录酶