ࡱ> ruq_ bjbjڿڿ *-b-b6!HZ( 8TG5(4444444$a7:4945|4402 'ӨeV1450G5l16:e:,2:244AG5:B : Probing Northern and Southern Blots This involves hybridising a radiolabelled probe (DNA or RNA) to your target (DNA or RNA) on a membrane. Probing has five basic steps: Pre-hybribisation (pre-hyb): The blot is incubated with hybridisation buffer at the hybrisiation temperature this lets blocking agents in the hybridisation buffer block non-specific binding sites. Hybridisation (hyb): The radioactive probe is added to the hybridisation buffer on the blot and incubated at hybridisation temperature overnight. Low stringency wash: After the probe is poured off, the blot is washed repeatedly with low stringency wash buffer (high salt) to remove unbound probe High stringency wash: The blot is washed 2-3 times with high stringency wash buffer (low salt) at high temperature to remove non-specifically bound probe. Exposure: The membrane is dried, wrapped in SaranWrap and exposed to a phosphorimage screen. The image is then read on a phosphorimager. There are many different types of probes and ways of doing the hybridisation. They all have different uses. Oligo probes these are very good for detecting high abundance targets by northern blot. On a good day they are very specific. They allow precise dissection of processing intermediates. They strip very easily which is very useful. Strand specific. Random prime probes The standard method for southern blots, not so good for northern blots. They strip reasonably well. They are not strand specific. Riboprobes the best probes for northern blots. Give a much higher signal to noise than random prime probes, and are strand specific. They strip badly, so if probing a blot sequentially, start with the weakest expected signal. Be aware that a weak signal from a new hybridisation may be un-stripped probe from the last hybridisation. Hybridisation in roller hybridisation bottles Hybridisation bottles are long, thin bottles that clip onto a rotisserie in a hyb oven. When using a rotisserie, always be sure to balance the bottles, including the liquid inside. Ensure that hyb bottles are fairly new and have decent seals. To put the membrane in the bottle, soak it in 0.1xSSC 0.1% SDS, roll it up with the DNA/RNA side inwards (though this doesnt seem to matter much). Drop into the hyb bottle then use a stripette to unroll it so that its flat against the glass. Push out any obvious air bubbles. This needs to be seen and practised. Two membranes can be hybridised in the same bottle, one facing the glass and the other facing the inside of the bottle. Put bottles in the oven so that the visible edge of the membrane is moving with the direction of rotation if your membrane rolls up into a thin cigar during hyb or washing then you have it the wrong way round. To clean the bottle, simply rinse with water, and give a wipe with a paper towel to remove residual counts. The lids are harder to clean: run water into the lid at high pressure (hopefully this will dislodge the rubber seal), and wipe inside with a paper towel. Do NOT attempt to prise the seal out this can damage the seal, leading to leaks during future hybridisation, which is very bad. It is often not possible to completely decontaminate hyb bottle lids, so we always treat them as if contaminated. If you are careful, the bottles, lids and seals last for many years. Random Primed DNA probe for southern blot The template should be a 100-1000bp PCR product (use ~1000bp for higher sensitivity eg. for a southern), and MUST be gel purified prior to use. Warm a falcon tube of Church hyb in 50C water bath until the SDS goes into solution. Put the blot in a hyb bottle and pre-hyb for ~1hr at 65(C with 7.5ml Church Hyb. Meanwhile, dilute 25ng probe DNA in 37(l H2O in a screw cap tube. Heat probe to 95C for 5 min then snap chill on ice for 5 min. Add: 10(l 5x home-made labelling buffer 1(l Klenow 5-3 exo- enzyme (NEB) 2(l (20(Ci) [(-32P]dCTP 3000Ci/mmol Pipette up and down to mix and incubate at 37( for 1-2 hours (better 2 hours). Flick a miniQuick Spin column (Roche) inverted and correct way up to get the sepharose to the bottom, remove lid then bottom cap (see product instructions) Place in a 2ml tube, spin at 1000g for 30s, discard tube. Place column in a screw cap tube then pipette probe carefully on to the centre of column Spin at 3000rpm for 4 minutes the probe is the flow through from the column Test incorporation by (briefly!) holding probe and column equal distances from a Geiger counter. Use tweezers for this. At least 30% of label should be incorporated. If the highest sensitivity is not required, not all the probe needs to be used once. Some probe can be stored at -20( until needed (shield this well as it is very hot) Boil probe for 5 minutes, snap chill on ice water for 2 min Add probe to the hyb bottle (drop it into the bottle while vertical so that it falls directly into the hybridisation buffer) and continue to rotate at 65( overnight Pour probe directly into the sink and wash down with plenty of water Pour ~50ml of 6xSSC in the bottle, close tightly and shake gently (make sure the membrane remains on the side of the bottle!), pour the wash into the sink Repeat the last step once Add 50ml of 6xSSC, return the bottle to the hyb oven and rotate at 65( for at least 15min. Make sure the bottle is balanced by another also containing 50ml liquid Pour off the wash buffer and add 50ml of pre-warmed (65() 0.5xSSC 0.1% SDS, return to hyb oven and rotate at 65( for 20min Repeat the last step once Pour off the remaining buffer, and use tweezers to pull the membrane out of the bottle into a box of room temperature 0.1x SSC 0.1% SDS Shake the blot gently for more than 5min-a few hours, then put the membrane on a paper towel and pat lightly to dry. Wrap the membrane in Saran wrap. Check the signal with a Geiger counter this provides a clue to exposure time Expose the membrane to a phosphorimager screen in a cassette. The screen should be blanked just before use as it will expose slowly with time High sensitivity random primed probe for a southern blot Pre-hyb the blot at 42( with UltraHyb (Ambion), after dissolving the buffer completely by incubating at 68( for >15min Follow the random primed southern blot protocol to make and denature the probe, then add to the hyb. After hybridiation, pour the used probe into a falcon tube and dispose as solid waste (Ultrahyb contains formamide and should not be poured down the sink). Perform washes as for southern blot protocol, BUT do all washes at 42( and use 0.1xSSC 0.1% SDS for the high stringency wash. See notes on background below as this is a common problem here. Low sensitivity probe for a northern blot For northern blot control probes that will give a very high signal it is often wise to use a random primed probe, which is easier to strip than a riboprobe. Follow the protocol for the random primed southern blot probe given above High sensitivity random primed probe for a northern blot Follow the High sensitivity random primed probe for a southern blot protocol above, but during the first 6xSSC wash, increase the oven temperature to 55(, then wash twice for 20 min with 0.2x SSC 0.1% SDS. Expose the blot. High sensitivity random primed probe for small RNAs (particularly siRNA) Use UltraHyb Oligo at 42( for hybridisation, and perform high stringency washes using 2xSSC 0.5% SDS. You will almost certainly need to scrub the membrane with a paper towel before exposing (see Removing residual background section later), but use 2xSSC 0.5% SDS instead of 0.1xSSC 0.1% SDS as this can strip the probe from siRNA targets. See note above about using less radiation to make the probe as that will probably reduce background a great deal. Removing residual background Sometimes membranes have a residual smeary background that interferes with your signal. This is more common with large membranes or when two membranes are probed in one bottle, or when the amount of target is low. Here are three solutions: Wash at room temperature in a plastic box on a rocker with copious 0.1xSSC 0.1%SDS for a few hours. If this fails, use a paper towel to scrub your membrane on both sides while it is under the SSC/SDS repeat until no more counts come off onto the paper towel (really! this will not damage the DNA/RNA or the probe but removes residual radioactive gunk). This is almost essential for blots probed with random primed probes in UltraHyb Oligo which seems particularly sticky. We have found that when using random-primed probes in UltraHyb for targets that are rare (so there is not much target at all on the membrane), unhybridised probe will tend to stick non-specifically across the membrane causing a very high background. This only applies to targets that would be below the normal detection threshold for normal Church Hyb. The effect is particularly bad with new radiation and at hybridisation temperatures of 50 or lower, and frustratingly gets worse as your probing improves! This can be partially removed by scrubbing the membrane with a wet paper towel as above but this is clearly unsatisfactory. The best solution we have found is to use less probe, which can be achieved by adding less radiation to the probing reaction (down to 0.2l fresh [(-32P]dCTP, scaled for half-life). Unfortunately this is something that varies probe-to-probe. Stripping membranes Wash with boiling 0.1xSSC 0.1% SDS in a plastic box with shaking, check residual signal with a Geiger counter and repeat if necessary Riboprobe for northern blot Templates for RNA probes are 100-500bp PCR products containing T7 sequence or (rarely) linearised plasmids containing the desired sequence and an RNA polymerase promoter. See the in vitro transcription protocol for more details. Pre-warm a bottle of UltraHyb (Ambion) to 68( until the SDS fully dissolves Put the blot in a hyb bottle and pre-hyb for 1hr at 68( with 7.5ml UltraHyb (use 10ml for a big membrane eg: 20x20cm) Synthesise a hot RNA probe using the separate in vitro transcription protocol. Flick a miniQuick Spin (Roche) column inverted and correct way up to get the sepharose to the bottom, remove top then bottom cap (see product instructions) Place in a tube, spin at 1000g for 30s, discard tube, place column in new tube Add 30(l water to probe, mix then pipette carefully on to the centre of matrix Spin at 1000g for 4 minutes Test incorporation by (briefly!) holding probe and column equal distances from Geiger counter. Use tweezers for this. At least 50% of label should be incorporated Add probe to the hyb bottle (drop it into the bottle while vertical so that it falls directly into the hybridisation buffer) and continue to rotate at 68( overnight Pour the probe into a 50ml Falcon tube and store in the freezer. Pour ~50ml of 6xSSC in the bottle, close tightly and shake gently (make sure the membrane remains on the side of the bottle!), pour the wash into the sink Repeat the last step once Add 50ml of 6xSSC, return the bottle to the hyb oven and rotate at 68( for at least 20min. Make sure the bottle is balanced by another also containing 50ml liquid Pour off the wash buffer and add 50ml of pre-warmed (68() 0.1xSSC 0.1% SDS, return to hyb oven and rotate at 68( for 20min Repeat the last step once3 Pour off the remaining buffer, and use tweezers to pull the membrane out of the bottle onto a paper towel. Pat lightly to dry. Wrap the membrane in Saran wrap. Check the signal with a Geiger counter this provides a clue to exposure time Expose the membrane to a phosphorimager screen in a cassette. The screen should be blanked just before use as it will expose slowly with time Oligo probe for northern blot Oligo probes are normally 20-34bp, 40% GC. Some probes work, some dont, Ive never been entirely sure why. Probes should not have a C at the 5 end (inhibits labelling), and currently work best if cleaned down a Qiagen Nucleotide Removal column before use. This has not always been the case and is presumably due to some contaminant in Sigmas manufacturing process at the time of writing. Pre-hyb blot for 1 hour in 10ml Oligo Hyb at 37( Mix 5pMoles oligo 1.5(l 10x T4 polynucleotide kinase buffer 1.5(l 100mM DTT Water to 12(l Then add 1(l T4 polynucleotide kinase 2(l [(-32P]ATP 6000Ci/mmol Incubate 30 minutes at 37( Clean probe through a column as for a riboprobe, BUT use a miniQuick Spin Oligo column (oligos stick to normal miniQuick spin columns) Add probe to pre-hyb and incubate overnight at 37( Store and reuse probe as for a riboprobe Rinse then wash 10min with 50ml 6xSSC, See riboprobe protocol for general washing and exposing directions Wash 10min with 50ml pre-heated 2xSSC 0.1% SDS at 37(, then 10min with 50ml room temperature 6xSSC. High sensitivity oligo probe for northern blot Use UltrHyb Oligo at 42( for hybridisation, and perform high stringency washes using 2xSSC 0.5% SDS. Better with longer oligos (>30nt) and 40-60% GC Solutions Oligo Hyb: 0.17Moles Na2HPO4 0.079Moles NaH2PO4 Moles not g given as hydration state of stocks varies widely 35g SDS 1ml 0.5M EDTA dH2O to 500ml and warm to dissolve pH should be ~7.2 Store at RT Church Hyb: Dissolve the ingredients given above for oligo hyb in 400ml water Dissolve 5g BSA in 100ml water Cool the oligo hyb to room temperature, then slowly add the BSA solution while stirring Store in aliquots at -20(, working aliquot at 4( Ultrahyb and UltraHyb Oligo purchased from Ambion then split into 4 aliquots and store at 4( Incubate all hybridisation buffers at 55-60( or 68 for Ultrahyb in a water bath for ~20min to re-disolve the SDS. 5x home-made labelling buffer (for random priming): Buy Random 9-mers from NEB (S1254S), dissolve in 33l water to 1mg/ml. 250l NEBuffer 2 12.5l 1mg/ml d(N)9 0.825l 100mM dATP 0.825l 100mM dGTP 0.825l 100mM dTTP water to 500l split into 100l aliquots and freeze  32P does not need to be particularly fresh we use up to 6 weeks old with no problem, older for high signals. The required exposure time obviously increases, but the output is similar.  If the membrane is too far down, add some 6xSSC, screw on the lid tightly and shake until the membrane comes loose and lodges close to the top of the bottle. Unscrew the lid and carefully pour off buffer be careful the blot does not end up in the sink, though it will not do much harm  Probe re-use: Pre-hyb the blot with the normal quantity of Ultrahyb. Defrost the probe by letting the falcon tube stand in a beaker of hot water for 20min then pour the probe into the hyb bottle. Probes can be re-used multiple times. Probe should be disposed to solid waste once no longer needed.  Use 0.1x SSC 0.1% SDS at 68( for these two washes to increase stringency if cross-hybridisation is a problem  Do not use too much oligo!  Washes for oligos need to be optimised increase the time and number of 2xSSC 0.1% SDS washes, and increase washing temperature to 42(      FILENAME Probing Blots v2.9#$%}v 7 s . /  5 *8?@Ja35 ޽޽ȽȽȽȽ{ph^Z5CJOJQJhkO5CJOJQJhw5CJOJQJhwhwCJOJQJhUCJOJQJh:CJOJQJhK5CJOJQJh5CJOJQJh#5CJOJQJh15CJOJQJh=X5CJOJQJh=^P5CJOJQJhWh=^Ph=X*$%s  7 . / ( ) @A45gd: 0^`0gdw 0^`0gd1 0^`0gd=Xgd=XgdC_ JO`()*BIY~pf[PEPPEh,35CJOJQJhv65CJOJQJhdL5CJOJQJhotCJOJQJhU7hot5CJOJQJh,3CJOJQJhU7CJOJQJhfCJOJQJhC_hC_CJOJQJhCJOJQJhCJOJQJh5CJOJQJh:5CJOJQJhm5CJOJQJhkO5CJOJQJh^Z5CJOJQJh]4F5CJOJQJ)*cd4Z[GH+gdf:?@AIJKLPQTVW]abcdpv{ȽȲޙәӎuj_Ƚhf5CJOJQJh[5CJOJQJh[hg15CJOJQJhkO5CJOJQJh&5CJOJQJhg15CJOJQJ jhg15CJOJQJhFT85CJOJQJh]4F5CJOJQJh,35CJOJQJh+cx5CJOJQJh[5CJOJQJh 5CJOJQJhH5CJOJQJ"øیvیvhۭہ jmhf5CJOJQJh=^P5CJOJQJhH5CJOJQJhb:5CJOJQJh9)H5CJOJQJhf5CJOJQJhf5CJOJQJh[5CJOJQJh[5CJOJQJh\L5CJH*OJQJh\L5CJOJQJhT+5CJOJQJ jmh=^P5CJOJQJ$"'-3467;<=BCDFJXYZ[u{Ⱥ~laaӤSHhK5CJOJQJ jh=^P5CJOJQJhb:5CJOJQJ"jh]4F0J5CJOJQJUhY5CJOJQJh=^P5CJH*OJQJ jah=^P5CJOJQJh=^P5CJOJQJhw,5CJOJQJ jmh=^P5CJOJQJh]4F5CJOJQJhf5CJOJQJhH5CJOJQJh\L5CJOJQJhv65CJOJQJSTWor*,ӽӲӧӜӲӑӆȲӆ{p{hl5CJOJQJh5CJOJQJhv65CJOJQJhf5CJOJQJhf5CJOJQJh]4F5CJOJQJh[5CJOJQJhF5CJOJQJhot5CJOJQJh=^P5CJOJQJhb:5CJOJQJhK5CJOJQJh 5CJOJQJ)+,{|^_@A[\{| ! gddL,BMz|&GH|6LRS_Ӄxm_Thg15CJOJQJ jhg15CJOJQJhB5CJOJQJh\L5CJOJQJhf5CJOJQJh=^P5CJOJQJ jhM5CJOJQJh5CJOJQJhM5CJOJQJhg[5CJOJQJhY5CJOJQJhot5CJOJQJhl5CJOJQJhPt5CJOJQJh?5CJOJQJ_`dj-?UZ\78op{'FIUsx齯齯ӽӒȇȇ|rhotCJOJQJhg15CJOJQJhv65CJOJQJ"jhi[0J5CJOJQJUh]4F5CJOJQJ jhB5CJOJQJhB5CJOJQJh55CJOJQJhi[5CJOJQJhdL5CJOJQJhY5CJOJQJh+cx5CJOJQJ'I J L!M!!! " "7"8""" #!#"###$#%#^#_#@$A$gdg[gdm   < = L!!!! " " "6"## #!#%#7#_#۷۬۬“ti_UKh,CJOJQJhmCJOJQJhCJOJQJh,5CJOJQJhg[5CJOJQJhg[CJOJQJhy5fCJOJQJhmh5CJOJQJh 5CJOJQJh]4F5CJOJQJh5CJOJQJh5CJOJQJ jhm5CJOJQJhm5CJOJQJhmhg[CJOJQJhmhmCJOJQJ_#k#####?$@$A$$$$$$$z%|%%%%%%Q&R&S&q&+'E'((&)')=)·~shh^hShShShh~W5CJOJQJhCJOJQJh5CJOJQJhGK5CJOJQJhw,5CJOJQJh 5CJOJQJ jhp15CJOJQJhu5CJOJQJhp1CJOJQJhp15CJOJQJhm5CJOJQJ jh,5CJOJQJh,5CJOJQJh h 5CJOJQJh 5CJOJQJ A$$$R&S&p&q&a'b''=)P-R-z-|-D.E.F.G.d.e.K/L///gddL & Fgdn & Fgdgdgdg[gdp1=)k*r***++++~,,,,,,P-x-F.G.H.L.Q.c.d.e....ۚ|odYNYh 5CJOJQJhdL5CJOJQJhC_5CJOJQJhC_hC_CJOJQJhJNCJOJQJh46CJOJQJhw[CJOJQJhm5CJOJQJhCJOJQJhh5CJH*OJQJ jah5CJOJQJhh5CJOJQJh5CJOJQJh~W5CJOJQJhh5CJOJQJ.... //// /L/X/u/x/y/////// 00000"0+02060?0G0_0`0T1U1`2Ӱӗӗ{m_ jmhdL5CJOJQJho.hdL5CJOJQJh[h+cx5CJOJQJ jh+cx5CJOJQJh+cx5CJOJQJ jhakX5CJOJQJh5CJOJQJhakX5CJOJQJhdL56CJOJQJh 5CJOJQJhKx5CJOJQJhdL5CJOJQJhJN5CJOJQJ#/00`0a000M1N11111_2`233H3I3333444 5!5<5=5gd gddLgd+cx`222233E3F3H3I3%4E4F4444445555:5;5<56666ųťۚ{k`VLh CJOJQJhCJOJQJh 5CJOJQJhZ`h 5CJH*OJQJhZ`5CJH*OJQJ"jhZ`0J5CJOJQJUhm5CJOJQJh+cxh+cx5CJOJQJ"jh+cx0J5CJOJQJUh+cx5CJOJQJhdL5CJOJQJ jh 5CJOJQJh5CJOJQJh 5CJOJQJ=555.6/66666e8f8888888889:9;9V9W999:gdC`gd 'gd 'gd 666667%7H7d8f8888888888888888888ʿʴzj[Rh5OJQJ jmh 'h '5OJQJjhC0J5OJQJUhw[5OJQJh 'h '5OJQJh '5OJQJh 'hg15CJOJQJ jhg15CJOJQJh5CJOJQJhg15CJOJQJhC5CJOJQJh 5CJOJQJh '5CJOJQJhC_5CJOJQJhCCJOJQJ88888889999 9!9$9%9&9(9-999:9;9<9T9U9W9]9{md[LAh$g5CJOJQJ jh 'h '5OJQJhC5OJQJh '5OJQJh 'h '5H*OJQJ jgh 'h '5OJQJ jmh 'h '5OJQJh 'h '5OJQJ jmh 'hC5OJQJh 'hC5OJQJhC5OJQJ jmhC5OJQJhw,5OJQJh 'h5OJQJh5OJQJ jmh5OJQJ]9c9j9k9m9w99:::=:C:S:X:]:b:d::::::: ; ;;;;@;W;X;;;;;ӺӺӝzoahh5CJOJQJh5CJOJQJ jh5CJOJQJh|5CJOJQJhCJOJQJh 5CJOJQJ"jhr~0J5CJOJQJUhC5CJOJQJ jhg15CJOJQJhg15CJOJQJh '5CJOJQJh$g5CJOJQJhC5CJOJQJ"::<:=:e:f:::;;?;@;;;;;<<U<_<o<<<<<=%=gddL^gd ^gd_);;;;;;;;;;;<<<<<T<U<X<s<t<<<<}=======ɾ}rg\N\N\g jhw,5CJOJQJhw,5CJOJQJhdL5CJOJQJh 5CJOJQJh465CJOJQJh5CJOJQJh_)5CJH*OJQJh_)h_)5CJH*OJQJh_)h_)5CJOJQJh_)5CJOJQJh25CJOJQJhg15CJOJQJhChCCJOJQJhCCJOJQJhw,CJOJQJ%=}===>>>>>???1?I?\?t????o@A~BBgdH @ ^@ `gdM\q^gdM\q ^`gdfgdr~gddL^gdr~==== > >:>;>O>>>>>>>>>>>>>>??????.?/?5?6?[?\?`?a?x?y???????ź{qjh]4F0JUhHhM\q5CJOJQJhHhH5CJOJQJhH5CJH*OJQJhM\q5CJOJQJhf5CJOJQJhH5CJOJQJhC5CJOJQJh5CJOJQJ jhw,5CJOJQJhw,5CJOJQJhr~5CJOJQJ+?????@@@"@n@o@p@AABBBB,C-CICJCCCCCCCCCCCCCCCCCCCC Ӷwh^Z5CJOJQJaJUh~W5CJOJQJaJ$h^Z5CJOJQJaJmHnHuhh^Z5CJOJQJaJ(jhh^Z5CJOJQJUaJjhnUhn jh^Zh^Zjh^Z0JU h]4Fh]4Fh h]4FH*h]4F,B,CICCCCCCCCCCCgdr~gdgdr~ Houseley lab  PAGE 9 hHhM\q5CJOJQJhnh^Zjh^Z0JU*h~W0JmHnHu* h^Z0Jjh^Z0JU(. A!"#$%  s2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ OJPJQJ_HmH nH sH tH H`H Normal5CJOJQJ_HmH sH tH DA`D Default Paragraph FontViV  Table Normal :V 44 la (k (No List 8>@8 Title$a$ CJ(OJQJ4@4 Header  9r 4 @4 Footer  9r .)@!.  Page Number:@2: K Footnote TextCJT/AT KFootnote Text Char5OJQJmH sH tH @&`Q@ KFootnote ReferenceH*PK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vc:E3v@P~Ds |w< XE*,/ 2; y #;& ::GGGJ ",__#=).`268]9;=?"$&'()+,./124678:<=A+A$/=5:%=B#%*-0359;> %;BDJ!8@0(  B S  ? cl~ z } ~ $'7:"%TXVY"!!H%Q%%%k&q&&&&&''i'r'''q)t),+/+++..j/m/5090}000000000021;1j1s1D2K2223344744444G5O5q5x5D6H6W6[6o6s668888L9O9V:\::::::::::;; $%EEVV &!'!(!(!k"r"""""##6:::::: ;;;;; $%EEVV &!'!(!(!k"r"""""##6;lD$B$Cf O?@ABDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`bcdefghjklmnopstwRoot Entry F+Өv@1TableC:WordDocument *SummaryInformation(aDocumentSummaryInformation8iMsoDataStore$Ө 'ӨAZ4P00YZ3IF==2$Ө 'ӨItem PropertiesUCompObj r   F Microsoft Word 97-2003 Document MSWordDocWord.Document.89q