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基因组DNA的号子

文章作者:生命科学 上传时间:2019-08-27

核心提示:We typically use 0.5ug of E. coli genomic DNA in a labeling reaction for each hybridization. The genomic DNA was fragmeWe typically use 0.5ug of E. coli genomic DNA in a labeling reaction for each hybridization. The genomic DNA was fragmented to 500 to 1000bps before labeling. The following protocol should produce enough labeled probe for 8 hybridizations.

核心提示:E.coliTotalRNALabelingProtocolforSpottedMicroarrayNote:Start with 20 mg

核心提示:PCR: We generally use 1-2 ul of starting paraffin microdissected DNA for each 50 ul DOP-PCR reaction.We assume thatPCR:

For labeling 4ug Genomic DNA:

E.coliTotalRNALabelingProtocolforSpottedMicroarray

We generally use 1-2 ul of starting paraffin microdissected DNA for each 50 ul DOP-PCR reaction.

皇家赌场号hj85,DNA Mix

Note:Start with 20 mg of total RNA for each labeling reaction.All solutions that can be filtered should be filtered.

We assume that about 1 ug of product is produced in each DOP-PCR reaction. The entire product is then nick translated as described below.

Genomic DNA

Cy dyes are light sensitive and should ALWAYS be handled in dim light.

Nick Translation of the PCR Product:

For the first CGH, our approach is to start with predefined test and reference labels, and then to do a replicate reaction based on the initial results.

For the first CGH, the sample DNA is nick translated with digoxigenin-dUTP and stained with anti-digoxigenin-TRITC. 45 ul of the nick translation product is used. The amount of the digoxigenin sample should be reduced to 35 ul if the PCR product size is larger than ~600 bp.

The reference DNA is directly labeled with FITC-dUTP .

We use PCR-amplified DNA from the MPE600 cell line for a positive control CGH. Since this is good quality fresh DNA, only 10 ul of the dig-labeled DNA is necessary.

1.9ug/ul

RNA Preparation

CGH Hybridization

2.1ul

  • If RNA is in ethanol, spin down 20 mg of RNA per reaction @ 14000 rpm for 20 min. at 4oC.
  • Pipette off supernatant and wash pellet with 100ml of 70% ETOH.
  • Spin 5 min. and remove supernatant without disturbing pellet
  • Air dry pellet 15-20 min at Room Temp .
  • Resuspend RNA pellet in 12.5 ml DEPC H2O

1) Reprecipitate DNA's

  • Add the following DNA's to a 1.5 ml centrifuge tube, mixing with pipet:20 ug Cot-1 DNA ~900 ng FITC labeled DNA ~200 ng Texas Red labeled DNA
  • Add 1/10th volume of 3M Na Acetate, mixing with pipet.
  • Add 2.5 X vol 100% EtOH to ppt DNA, vortex gently.
  • Spin 30 mins at 14K rpm, 4 C.
  • Decant supernatant; blot dry, being careful to avoid DNA pellet.
  • Add 10 ul of MM1/H20 mix .
  • Carefully dissolve with pipet, and gently vortex.
  • Quickly spin to bring volume to bottom of tube.

Random Hexamer

RNA

5mg/ml

12.5ul

The Inverse Hybridization

Based on the initial CGH hybridization using digoxigenin labeling, and the PCR product size, the following algorithm is used to decide on the second hybridization:

If the initial CGH using the digoxigenin labeled DNA was bad, the PCR is repeated using more DNA, and if the CGH still fails, a second microdissection is considered.

If the probe size was good and the CGH using digoxigenin labeled probe was bright and uniform, then the second inverse reaction uses sample DNA directly labeled with FITC-dUTP and reference DNA directly labeled with Texas Red dUTP .

If the CGH using the Digoxigenin labeled DNA looked good, but the probe size wasn't ideal then the test sample is labeled with Digoxigenin and stained with anti-digoxigenin-TRITC , and the reference DNA is labeled with biotin and stained with FITC-Avidin .

1ul

Random Hexamer

H2O

基因组DNA的号子。5mg/ml

14.9

1ul

Total

Labeling Control

20ul

1ul

Heat to 95C for 5min, place on ice for 5min

Total

Labeling

17ul

DNA Mix

Heat to RNA to 70 oC 5 min, ice 2 min, pulse spin

20ul

Labeling

dAGC

Prepare labeling mix .

5mM each

1X labeling mix

First Strand Buffer

5ul

5x

EcoPol Buffer

8ul

10x

DTT

5ul

0.1M

CyDye-dUTP

4ul

1mM

dNTPs**

2ul

10x

H2O

4ul

17ul

RNAsin

Klenow Fragment

1ul

50u/ul

Total

1ul

17ul

Total

  • To the RNA/hexamer mix add 17.0 ml of labeling mix and incubate 10min at RT
  • Add 1.5 ml of appropriate CyDye dUTP followed by 1.5 ml SSII reverse transcriptase, mix well by tapping and pulse spin
  • Incubate 1hr at 42 oC in the dark
  • Add an additional 1.5 ml SSII reverse transcriptase, tap, pulse spin and continue incubation 1hr
  • Degrade RNA by addition of 2 ml 1N NaOH, vortex, pulse spin and incubate 15 min at 65 oC
  • Neutralize by addition of 2 ml 1N HCl, vortex and pulse spin

20ul

Clean up Labeled Probes

Incubate at 37°C for 3.5 hours

  • Prewash Microcon-30 microfilter by adding 450ml miliQ H2O and spinning for 10 min. @ 12,000 RPM.
  • Add 450ml miliQ H2O to each of the probe samples . Mix thoroughly by pipetting up and down. Transfer samples to separate Microcon-30 microfilters.
  • Spin at 12,000 RPM in microfuge for 10 minutes or until 20-40 ml remains in the filter.
  • Add 450 ml miliQ H2O to the probe and gently mix by pipetting up and down. Be careful not to touch the filter at the bottom of the filtration unit.
  • Spin 10 minutes at 12,000 RPM
  • Repeat step 4 , spin 12min to get smaller volume.
  • Invert column into a fresh tube and spin 1 minute at maximum speed to recover probe. Carefully measure recovered probe volume if necessary.
  • Transfer recovered probe to the appropriate partner probe.

Add 2.5ul 0.5M EDTA to stop reaction

Note: Probes can be combined after the first wash step.Probe can be stored at 4C or -20C in dark for further purpose.

Clean up Labeled Probes

Reagents and Suppliers

  • Prewash Microcon-30 microfilter by adding 450ml miliQ H2O and spinning for 10 min. @ 12,000 RPM.
  • Add 450ml miliQ H2O to each of the probe samples . Mix thoroughly by pipetting up and down. Transfer samples to separate Microcon-30 microfilters.
  • Spin at 12,000 RPM in microfuge for 10 minutes or until 20-40 ml remains in the filter.
  • Add 450 ml miliQ H2O to the probe and gently mix by pipetting up and down. Be careful not to touch the filter at the bottom of the filtration unit.
  • Spin 10 minutes at 12,000 RPM.
  • Repeat step 4 , spin 12min to get smaller volume.
  • Invert column into a fresh tube and spin 1 minute at maximum speed to recover probe. Carefully measure recovered probe volume if necessary.
Cy3-dUTP 1mM Perkinelmer NEL578
Cy5-dUTP 1mM Perkinelmer NEL579
SuperScript II 200U/ul Invitrogen 18064-014
RNAsin 20-40U/ul Promega N2515
100 mM dNTP set** 10X Amersham 27-2035-01
pd6 Sodium Salt 50U Amersham 27-2166-01
Microcon YM-30 column Amicon 42410

Other reagents: 20X SSC, TE pH7.4, 10% SDS, 500 mM EDTA, 1M NaOH, 1M Tris-HCl pH7.5, sterile dH2O and DEPC H2O* comes lyophilized, must be resuspended at specified concentration.

Probe can be stored at 4°C or -20°C in dark for further use.

Reagents and Suppliers

Cy3-dUTP 1mM Perkinelmer NEL578
Cy5-dUTP 1mM Perkinelmer NEL579
Klenow Fragment 50U/ul NEB M0210M
100 mM dNTP set* 10X Amersham 27-2035-01
pd6 Sodium Salt 50U Amersham 27-2166-01
Microcon YM-30 column Amicon 42410

*for 10X stock: 5 mM each of dA, dG, dC.

**for 10X stock: 5 mM each of dA, dG, dC and 2 mM of dT in DEPC H2O

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