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Home Protocols In-situ hybridization Probe construction for in-situ hybridizations

Probe construction for in-situ hybridizations

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Version: Shini/Matt/Aubrie 01/28/09 (to be completed)

 

1. PCR amplification of target sequence from plasmids (TOPO-pCRII)

 

For each probe, set up:

 

1x (25 uL rxn)

PCR-grade H2O

17.375 uL

10 x Ex Taq PCR buffer

2.5 uL

dNTPs (2.5 mM)

2 uL

Primer M13_fw (10 uM)

1 uL

Primer M13_rv (10 uM)

1 uL

Ex Taq polymerase (5U/uL)

0.125 uL

Plasmid DNA (~20 ng/uL)

1 uL

 

M13 Forward (-20)       5´-GTAAAACGACGGCCAG-3’        (Tm:51)

M13 Reverse             5´-CAGGAAACAGCTATGAC-3’            (Tm:50)

 

Run PCR

Hot-start:             94°C – 2’

30 cycles:              94°C – 30”

                            50°C – 30”

                        72°C – 30”  (extension time depends on probe size)

1 cycle:            72°C – 8’

For ever:            4°C

 

Check PCR products

Run out 5 uL on a 1% agarose gel.

 

Clean up PCR products and nanodrop

Use the QIAquick PCR Purification Kit, elute in MiniElute columns:

Elute in 10 uL water

Nanodrop sample

 

Making the antisense (or sense-) probe

  • Make the (anti)sense probe
  • Use the following reaction for a total of 10 uL
  • 1 uL T7 RNA polymerase (or SP6)
  • Roche, #

1 uL DIG DNA labeling

  • Roche, #11727073910

1 uL 10X Buffer  (Buffer is the same for T7 or SP6)

  • Roche, #11465384001
  • Be sure to dissolve all the precipitate before using, i.e., vortex

0.25 uL RNasin (final 10 U)

3 uL PCR product (final 500 ng)

 

 

 

1X (10 uL rxn)

RNase-free water

3.75 uL

10X Buffer

1 uL

DIG RNA labeling mix

1 uL

RNasin

0.25 uL (final 10 U)

Template DNA

3 uL  (final 500 ng)

T7/SP6

1 uL

 

 

  • Incubate for 3.5 hours at 37 °C
  • Add 1 uL of DNase I and incubate for 15 min at 37 °C
  • place on ice

 

Precipitate RNA according to: http://www.ambion.com/techlib/tb/tb_160.html

 

·      add 29 uL water

·      add 10 uL LiCl2 (12 M)

·      chilled 30 min -20°C

·      centrifuge for 30 min 16,000 x g at 4°C

·      remove supernatant by aspiration

·      1 mL 70% EtOH

·      centrifuge for 10 min 16,000 x g at 4°C

·      remove supernatant and dry pellet for 10 min

·      resuspend pellet in 22.5 µL of Nuclease-free water.

use 1 uL for nanodrop

use 1.5 uL for RNA-gel

      pour 1% agarose gel

mix:

      RNA-sample      1.5   uL

     10x loading dye   1    uL

      EtBr (1mg/mL)      1    uL

     Formamide         6.5    uL

      final vol.       10   uL

      denature RNA for 5 min at 65°C

      place on ice for 5 min

      run out sample (at max. 5V/cm)

  • add 80 uL hybridization buffer and store probe until use

 

 

 

 

Materials

 

Takara Ex polymerase

Item: TaKaRa Ex Taq

Company: Takara Bio Inc.

Cat. #: RR001A (250 U)

 

Vector: pCRII-TOPO

Item: TOPO TA Cloning Kit Dual Promoter (20 rxns)

Company: Invitrogen

Cat. #: 45-0640

 

PCR-cleanup kit

Item: QIAquick PCR purification kit

Company: Qiagen

Cat. #: 28106 (250 rxns)

 

MiniElute kit

Item: MiniElute Reaction cleanup kit

Company: Qiagen

Cat. #: 28206 (250 rxns)

 

10X Buffer

Item: 10X Buffer

Company: Roche

Cat. #: 11465384001

 

DIG RNA labeling mix

Item: DIG RNA labeling mix

Company: Roche

Cat. #: 11277073910

 

T7 RNA polymerase

Item: T7 RNA polymerase

Company: Roche

Cat. #: 10881767001

 

SP6 RNA polymerase

Item: SP6 RNA polymerase

Company: Roche

Cat. #: 10810274001