TMR 2.

3 Practical: molecular biology methods

Protocol for Plasmid isolation: Mini preparation

1. 2. 3. 4.

We will start from step 5 Prepare 13 ml tube with 5 ml !" co#tai#i#g appropriate a#tibiotic $ampicilli# co#ce#tratio# ca# be up to %&&'g(ml). Pic* up the colo#y with sterile yellow pipette tip" drop the tip i# the tube +ha*e your tubes at the rotary sha*er at 3&&,%&& rpm at 3-. /. 0igher temperatures $up to %2. /) ca# be used for some plasmids a#d 1.coli strai#s. 2#cubate for 13 h $Most of the cultures will show sufficie#t de#sity after 3,1&h i#cubatio#).

5. Tra#sfer 1.5 ml of culture i#to a 1.5 ml tube a#d ce#trifuge i# a table top ce#trifuge at 3&&& rpm for 5 mi#. 4. 5iscard the super#ata#t. 7. 2f the pellet is small repeat steps 5 a#d 4. 8. 6dd 1&& 'l of P1 buffer $7iage#) a#d 8orte9 u#til the pellet is completely resuspe#ded. 9. 6dd 1&& 'l of P2 buffer $7iage#) a#d mi9 by sha*i#g i# upside,dow# positio#" i#cubate for 2,5 mi#. 10. 6dd 1&& 'l of P3 !uffer $7iage#) a#d mi9 8ery carefully. 11. /e#trifuge at 13&&& rpm for 15 mi# %0/. While ce#trifugi#g prepare a set of fresh 1.5 ml tubes. 12. Tra#sfer the super#ata#t i#to a fresh tube" try #ot to ta*e debris. 13. 6dd -5& 'l of 1&&: 1t;0" 8orte9 briefly. 14. /e#trifuge 2& mi# at 13&&& rpm" %0/ 15. 5iscard the super#ata#t a#d wash 5<6 Pellet with 5&& 'l of -&: 1tha#ol. 16. /e#trifuge 5 mi# at 13&&& rpm. 17. 5iscard the super#ata#t" let the pellet dry. 18. 5issol8e the 5<6 pellet i# 3& 'l of dest 02;.

TMR 2.3 Practical: molecular biology methods

Restriction enzyme digestion

Attention! All the enzymes must be kept on ice, after removing out of the 20 0C freezer 5igestio# of 5<6 with 2 or more e#=ymes: use simulta#eous digestio# i# a# appropriate buffer 2f you ca# fi#d a buffer i# which all e#=ymes ha8e sufficie#t acti8ity $usually #ot lower tha# 5&:)" you ca# set your digestio# with all e#=ymes simulta#eously. 2t is importa#t that the total 8olume of e#=ymes you add to your reactio# is #ot more tha# 1&: of the total reactio# 8olume. The reaso# for this is that some e#=ymes ha8e star$>) acti8ity whe# the co#ce#tratio# of glycerol i# the reactio# e9ceeds 5:. 19ample of digestio# reactio# for plasmid 5<6 Plasmid 5<6 !uffer $1&9) 1#=yme 1 $1&?('l) 1#=yme 2 $1&?('l) 02; ?p to 1 'g 2 'l 1'l 1 'l To 2&'l

2# our e9perime#t we will use: Plasmid 5<6 !uffer @@@@@@@@ $1&9) 1#=yme 1" @@@@@@@ 1#=yme 2" @@@@@@@ 02; 3 'l 2 'l 1 'l 1 'l 13 'l

To prepare this reactio# you first pipet together 5<6" buffer a#d water" 8orte9 the mi9ture a#d add the e#=yme. Mi9 by pipetti#g a#d i#cubate at least 1 h at 3-./ i# a Thermobloc*.

TMR 2.3 Practical: molecular biology methods

Isolation of genomic DNA from animal cells

1. Pipet 20 l IA!"N Protease #or proteinase $% into t&e 'ottom of a 1.( ml microcentrif)ge t)'e. 2. Add 200 l sample to t&e microcentrif)ge t)'e* )se )p to 1 + 10 , -"$2./ cells in 200 l P01. /. Add 200 l 0)ffer A2 to t&e sample. Mi+ 'y p)lse34orte+ing for 1( s. 2# order to e#sure efficie#t lysis" it is esse#tial that the sample a#d !uffer 6 are mi9ed thoroughly to yield a homoge#eous solutio#. Note: 5o #ot add 726A1< Protease or protei#ase B directly to !uffer 6 . 5. Inc)'ate at (,67 for 10 min. 5<6 yield reaches a ma9imum after lysis for 1& mi# at 54./. o#ger i#cubatio# time ha8e #o effect o# yield or Cuality of the purified 5<6. (. 0riefly centrif)ge t&e 1.( ml microcentrif)ge t)'e to remo4e drops from t&e lid. ,. Add 200 l et&anol #.,81009% to t&e sample* and mi+ again 'y p)lse3 4orte+ing for 1( s. After mi+ing* 'riefly centrif)ge t&e 1.( ml microcentrif)ge t)'e to remo4e drops from t&e inside of t&e lid. :. 7aref)lly apply t&e mi+t)re from step , to t&e IAamp Mini spin col)mn #in a 2 ml collection t)'e% ;it&o)t ;etting t&e rim. 7lose t&e cap* and centrif)ge at ,000 + g #<000 rpm% for 1 min. Place t&e IAamp Mini spin col)mn in a clean 2 ml collection t)'e #pro4ided%* and discard t&e t)'e containing t&e filtrate. /lose each spi# colum# i# order to a8oid aerosol formatio# duri#g ce#trifugatio#. /e#trifugatio# at full speed will #ot affect the yield or purity of the 5<6. 2f the lysate has #ot completely passed through the colum# after ce#trifugatio#" ce#trifuge agai# at higher speed u#til the 726amp Mi#i spi# colum# is empty. Note: Whe# prepari#g 5<6 from buffy coat or lymphocytes" ce#trifugatio# at full speed is recomme#ded to a8oid cloggi#g. <. 7aref)lly open t&e IAamp Mini spin col)mn and add (00 l 0)ffer A=1 ;it&o)t ;etting t&e rim. 7lose t&e cap and centrif)ge at ,000 + g #<000 rpm% for 1 min. Place t&e IAamp Mini spin col)mn in a clean 2 ml collection t)'e #pro4ided%* and discard t&e collection t)'e containing t&e filtrate. .. 7aref)lly open t&e IAamp Mini spin col)mn and add (00 l 0)ffer A=2 ;it&o)t ;etting t&e rim. 7lose t&e cap and centrif)ge at f)ll speed #20*000 + g> 15*000 rpm% for / min. 10. Recommended: Place t&e IAamp Mini spin col)mn in a ne; 2 ml collection t)'e #not pro4ided% and discard t&e old collection t)'e ;it& t&e filtrate. 7entrif)ge at f)ll speed for 1 min. This step helps to elimi#ate the cha#ce of possible !uffer 6W2 carryo8er.

TMR 2.3 Practical: molecular biology methods

11. Place t&e IAamp Mini spin col)mn in a clean 1.( ml microcentrif)ge t)'e #not pro4ided%* and discard t&e collection t)'e containing t&e filtrate. 7aref)lly open t&e IAamp Mini spin col)mn and add (0 l 0)ffer A" or distilled ;ater. Inc)'ate at room temperat)re #1(82(67% for ( min* and t&en centrif)ge at ,000 + g #<000 rpm% for 1 min. 2#cubati#g the 726amp Mi#i spi# colum# loaded with !uffer 61 or water for 5 mi# at room temperature before ce#trifugatio# ge#erally i#creases 5<6 yield.

TMR 2.3 Practical: molecular biology methods

Isolation of ?otal RNA from Animal 7ells

1. 5etermi#e the #umber of cells. Pellet the appropriate #umber of cells by ce#trifugatio# at 5&& 9 g for 5 mi#. 6spirate the super#ata#t. 2. 5isrupt cells $ do #ot use more tha# 1 9 1&- cells) with ?R$ 2ysis ')ffer @ptional: As a preparation step add 20Al of 2.mercaptoet&anol per 1ml of ?R$ 2ysis 0)ffer in order to ac&ie4e ;orBing sol)tion. ?&is mi+t)re can 'e stored 1 ;eeB at room temperat)re. D 5 9 1&4 use 35& 'l !uffer 4 5 9 1& E 1 9 1& use -&& 'l !uffer 3. 0omoge#i=e cells with a syri#ge a#d #eedle by passi#g the lysate through a #arrow #eedle $1F,21 gauge) 5,1& times Note: Incomplete &omogenization of t&e sample ;ill ca)se lo;er yields and clogging of t&e col)mn. %. 6dd a# eCual 8olume $35& 'l or -&& 'l) of -& : 1tha#ol to the lysate a#d mi9 thoroughly by 8orte9i#g. 5o #ot ce#trifuge. 5. 6pply the sample to a 0i!i#d R<6 spi# colum# placed i#to a 2 ml collectio# tube. /e#trifuge at 1& &&& 9 g for 3& sec at room temperature. 5iscard flow,through. Note: ?&e ma+im)m capacity of t&e -i0ind RNA spin col)mn is <00Al 4. Wash the 0i!i#d R<6 spi# colum# with R<6 Wash !uffer 2 by pipetti#g 5&&'l directly o#to the spi# colum#. /e#trifuge at 1& &&& 9 g for 3& to 4& seco#ds a#d discard the flow,through. -. 6dd 5&& 'l of R<6 Wash !uffer 22 diluted with absolute etha#ol. /e#trifuge at 1& &&& 9 g for 3& to 4& seco#ds at RT. 5iscard flow,through. 3. With the empty 2 ml collectio# tube" ce#trifuge the 0i!i#d R<6 spi# colum# for 2 mi#utes at ma9imum speed to completely dry the colum#. F. 1lutio# of R<6: Tra#sfer the colum# i#to a clea# 1"5 ml ce#trifuge tube a#d elute the R<6 with %&,-& 'l of 51P/ treated water. Ma*e sure that you add the water directly o#to the 8olume matri9. /e#trifuge for 1 mi#ute at 1& &&& 9 g Note: Pre3&eating t&e ;ater to :0 07 'efore adding of to t&e col)mn* and inc)'ating t&e col)mn for ( min. at room temperat)re 'efore centrif)gation may increase yields

TMR 2.3 Practical: molecular biology methods

Real3time R?3P7R 1. Prepare 2 master mi9es for ge#e of i#terest $@@@@) a#d house *eepi#g $A6P5) ge#e for 2 samples each. Master mi9 Primer mi9 Water for 1 sample 5.& 'l &.5 'l %.& 'l for 2 samples 1& 'l 1 'l 3 'l

2. Gorte9" spi# dow# 3. %. 5. 4. -. 3. 6dd 1 'l c5<6 to each master mi9. Gorte9" spi# dow#. 5istribute master mi9 with c5<6 o# the plate F.5 'l per well. Place the plate i#to the thermocycler 2#itial de#aturi#g F5./ for 1& mi# 5& cycles: de#aturi#g F5./ 15 sec" a##eali#g(elo#gatio# 4&./ 1 mi#" collect data at the e#d of the step 29

TMR 2.3 Practical: molecular biology methods

Imm)no&istoc&emistry of spleen #-)man%

Pero9idase $61/) $5a*o/ytomatio#" cat.#o. B%&&%" B%&&5" B%&&3" B%&&F) 6tte#tio#:

?se a humid chamber for i#cubatio# stepsH Perform the wash steps i# glass,cu8ettes This protocol is usable for cryo,sectio#s +ectio#s were fi9ed with PI6" the# fro=e# at ,3&./ u#til use

1. Remo8e the slides out of ,3&./. 6ir dry at RT for about 15 mi#. 2. Ma*e a circle arou#d the sectio#s with the 5a*o,pe# $5a*o,Pe# from 5a*o/ytomatio#" cat.#o. +2&&2) 3. Wash: 1 9 P!+19(&"1:Twee# 2&" 3& sec. $P!+ 5ulbecco Powder" w(o /a2J" Mg2J" !iochrom" cat.#o. 132,5&)" $Twee#2& "+igma" cat.#o. P,-F%F). %. 6pply 5&' of Pero9idase !loc* $&.3: Pero9idase i# P!+19)" i#cubate 5 mi#. at RT 5. Wash: 1 9 P!+19(&"1:Twee# 2&" 3& sec. 4. Primary a#tibodies: sectio# 1:a3sta'ilin31 ra''it anti'ody R11 $dilutio# 1:3&&) sectio# 2 a3ra''it Ig! $wor*i#g co#ce#tratio# 2 'g(mlK dilutio# of commercial stoc* 1:-&&) Primary a#tibody has to be diluted i# 6#tibody 5ilue#t $5a*o/ytomatio#" /hemMate" cat.#o. +2&22). Ta*e 5&' of the a#tibody,co#tai#i#g solutio# per sectio#(circle. Working concentration of primary antibody will be prepared by Amanda as a master mix for all students since it I impossible to make 1:800 dilution in 50 l -. 2#cubate 3& mi#. at RT. 3. Wash: 3 9 5 mi#. P!+19(&"1:Twee# 2&. F. +eco#dary a#tibody: a3ra''it Ig!3-RP #dilutio# 1:%&&) i# 6#tibody 5ilue#t. Working concentration of primary antibody will be prepared by Amanda as a master mix for all students 6pply 5&' " i#cubate %5 mi#. at RT. 1&. Wash: 3 9 5mi#. i# P!+19(&"1:Twee# 2&. 11. 5e8elopme#t: 6pply ready,to,use 61/J substrate,chromoge# solutio# 12. 2#cubate 15 mi#. 13. ;bser8e the color de8elopme#t u#der a microscope to pre8e#t bac*grou#d stai#i#gHHH 1%. 6fter color de8elopme#t put the slides bac* i# P!+19(&"1: Twee#2&

TMR 2.3 Practical: molecular biology methods

15. /ou#terstai#: use MayerLs 0aemalau# for microscopy $Merc*" cat.#o. 1&F2%F). 14. 2#cubate 3& sec. at RT. 1-. Wash: ru# tap water i#to cu8ette u#til water becomes clear 13. Mou#t with 5a*o/ytomatio# aCueous mou#ti#g medium $5a*o/ytomatio#" cat. #o. +3&25): 2 sectio#s per slide , use 1 drop per sectio# a#d use a co8erslip with the si=e 2%94& mm 1F. +torage: 6ir dry the mou#ti#g medium. 6fter docume#tatio# store slides i# special Mslide bo9esM at RT. ! " oom temperature

TMR 2.3 Practical: molecular biology methods

?ranslational Medical Researc& Mod)l 2./ CA71 1taining of P0M7s #Perip&eral 0lood Monon)clear 7ells%
Prepare si#gle cell suspe#sio# of cells $591& 4) to be labeled $eg. peripheral blood mo#o#uclear cells of whole blood)" well washed" a#d resuspe#ded i# NI6/+ bufferM $phosphate buffered sali#e OP!+P co#tai#i#g 5: I/+). Tra#sfer cells i#to a 1"5 ml 1ppe#dorf Tube. Wash o#ce with 5&& 'l I6/+ !uffer" ce#trifuge at 12&&rpm" 5 mi#" % o/. 5iscard the super#ata#t. Resuspe#d the Pellet with 1&& 'l I6/+ !uffer. +amples to prepare per group: a) 2 'l /51% I2T/ 6b b) 2 'l /514 6P/ 6b c) 2 'l /51% I2T/ 6b J /514 6P/ 6b d) 2 'l 2sotype 1 I2T/ e) 2 'l 2sotype 2 6P/ f) 2 'l 2sotype 1 J 2 'l 2sotype 2 g) <o#,labelled cells 2#cubate for 3& mi#utes o# ice" *eep the samples i# the dar*. Wash with 5&&'l I6/+ !uffer" ce#trifuge at 12&&rpm" 5 mi#" % o/ 5iscard the super#ata#t. Resuspe#d the pellet i# 5&& 'l I6/+ !uffer or for Ii9atio# i# 2&&'l 2: PI6 Tra#sfer the cell suspe#sio# i#to a 5ml rou#d bottom I6/+ Tube. Beep the cells o# ice or i# the fridge