5.@ DNA and RNA gel-blot and RT-PCR analyses

5.1 DNA gel-blot analysis

 
Minoru Kubo, Yoshikatsu Sato
and Tomoaki Nishiyama

 

Introduction

DNA gel blot analysis is one of the most effective methods to estimate transgenic P. patens in which transgenes are integrated in the genome. To eliminate the risk of mistaking other effects such as genome rearrangement and multiple-insertion for transgene effects, we recommend selecting the transgenic lines that have simple and predictable insertion of transgenes.

Genomic DNA is digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through an agarose gel. The DNA is then denatured in situ and transferred from the gel to a solid support (usually nylon membrane). The relative positions of the DNA fragments are preserved during their transfer to the filter. The DNA attached to the filter is hybridized to radiolabeled or enzyme-labeled DNA, and autoradiography or chemiluminescenceis used to locate the positions of bands complementary to the probe. @

 

1) Preparation of the DNA-blotted membrane

Materials

E 3 -5 µg genomic DNA

E Appropriate restriction enzyme

E Ethanol

E 3 M CH3COONa pH 5.2

E TAE

E SeaKem GTG Agarose

E 0.25 N HCl

E Denaturing solution (0.4 M NaOH, 1.5 M NaCl)@

E Alkaline transfer buffer (8 mM NaOH, 1.5 M NaCl)

E 2x SSC

E Nylon membrane Hybond N+ (Amersham)

E Whatmann 3MM paper

E Paper towel

E Electrophoresis apparatus (Nippon eido NB1011: 140 mm x 150 mm gel, BIO craft BE520: 135mm x 150 mm gel)

E Power supply

E Oven

 

Procedure

1. Digest 3 µg of genomic DNA with an appropriate restriction enzyme. We usually use 15-20 units per µg DNA and incubate the solution at 37˚C overnight.

2. To purify the digested DNA, precipitate with ethanol.

3. Add 17 µl of TE, keep a few minutes on bench, and completely dissolve by vortexing.@ Be careful not to miss the DNA pellet which often sticks to the inner tube wall during vortexing.

4. (Check 1µl of digested DNA by electrophoresis)

5. Add 1.6 µl of 10x dye and mix.

6. Apply each sample and DNA ladder marker to agarose-gel (0.7% gel) and perform electrophoresis to separate the DNA fragments (Constant Voltage = 50 V for 30 min and 20 V for 12-16 hrs).

7. After electrophoresis, stain the gel with EtBr and take a photo with a scale.

8. For depurination, incubate the gel in 0.25 N HCl for 10 min at room temperature.

9. Rince with H2O once

10. For denaturation, incubate the gel in denaturing solution for 1 hr.

11. After denaturation, transfer the DNA to a nylon membrane Hybond N+ (Amersham Biotech) by a downward method (Chomczynski (1992) Anal. Biochem. 201:134-139) 1-2hr

12. Rince the membrane with 2x SSC.@

13. Dry at 80˚C for 60 min.

 

[Key points]

E BglII, EcoRI, EcoRV, EcoT22I, EcoT14I, HincII, HindIII and BamHI work well for full digestion of P. patens genomic DNA.

 

2) Hybridization

(1) Using non-RI system.

Please refer to the AlkPhos direct manual (Amersham Biotech: RPN3690). In this section, a protocol for hybridization and detection is described.

 

Materials

E Hybridization buffer (refer to the manual)

E Primary wash buffer (refer to the manual)

E Secondary wash buffer (refer to the manual)

E CDPstar (Amersham)

 

Procedure

1. Pre-heat buffer and hybridization bottles at 55˚C.

2. Place a membrane in a bottle and add 10ml of hybridization buffer in the bottle.

3. Incubate at 55˚C for at least 20 min.

4. Add probe solution in the bottle.

5. Incubate at 55˚C overnight (14-16 hrs).

6. After hybridization, wash the membrane in first wash buffer at 55˚C for 10 min, twice.

7. Wash the membrane in secondary wash buffer for 5 min, twice.

 

 

8. Drop 1-3 ml of CDPstar on the membrane and incubate for 5 min.

9. Discard excess CDPstar from the membrane, and wrap it with Saran wrap.

10. Expose the membrane by LAS3000 (high-mode, 2-3hr: super-mode, 1-2hr) or by X-ray film for 1 hr.

@

(2) Using RI-labeled probes

This protocol is that of Church and Gilbert (1981).

 

Materials

E Template DNA (>25 ng)

E High Prime DNA Labeling kit (Roche: cat.no. 1 585 592)

E [ƒż-32P] dCTP (3000Ci/mmol)

E TE (10 mM Tris-HCl(pH8.0), 1 mM EDTA)

E Sephadex G50

E 1 M ChurchNaPi buffer (0.5M NaH2PO4, adjusted to pH7.2 with H3PO4)

E Hybridization buffer

Stock solution

/50 ml

Final conc.

1M Church NaPi buffer

25 ml

0.5 M

0.5 M EDTA pH8.0

100 µl

1 mM

SDS (powder)

3.5 g

7% (w/v)

H2O

Up to 50 mL

 

E Wash solution

Stock solution

/1000 ml

Final conc.

1M Church NaPi buffer

40 ml

40 mM

SDS (powder)

10 g

10% (w/v)

H2O

960 mL

 

 

E Hybridization oven

E Hybridization bottle

E Sandwich box

E Water bath

E Heat block

E Incubator (37˚C)

 

Procedure

Probe preparation

1. Add 12.5 ng template DNA and H2O to a final volume of 5.5 µL in an eppendorf tube.

2. Denature the DNA by heating in a boiling water bath or heat block and chilling quickly in an ice bath.

3. Add 2 µL of High Prime solution and 2.5 µL of 32P-dCTP solution to denatured DNA solution.

4. Incubate for 10 min at 37˚C.

5. Stop the reaction by adding 2 µL of 0.2 M EDTA (pH8.0) and/or by heating to 65˚C for 10 min.

6. De-salt the labeled probe and remove unincorporated dCTP by applying to Sephadex G50.

Hybridization, wash and detection

1) Put the membrane in a hybridization bottle.

2) Add 5~10 ml of hybridization buffer to the bottle.

3) Pre-hybridize the membrane in Church Buffer for 30 min at 65˚C.

4) If the probe is double-stranded, boil 100 µl of probe for 5 min to denature the DNA and immediately transfer it to an ice bath to cool for 5 min.

5) Add the denatured probe to the pre-hybridization buffer, being careful not to add the probe directly onto the membrane.

6) Hybridize overnight at 65˚C.

7) Remove the hybridization solution and rinse the membrane in wash buffer.

8) Transfer the membrane to 300 ml wash buffer. Incubate for 20 min at 65˚C. Replace with fresh wash buffer and incubate for 20 min at 65˚C.

9) Wrap the moist (but not dripping wet) membrane in Saran wrap. Do not let the membrane dry out if it is to be either reprobed or washed further later.

10) Place in an x-ray cassette. Cover the membrane with a sheet of x-ray film and one intensifying screen. Store at -70 C overnight.

11) Develop the x-ray film and label for marker and for each lane. @Store the wrapped membranes at –20˚C.

 

 

References

(1) Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81: 1991-1995.