PCR: Principles of PCR BonesLab (degenerate PCR) Boehringer (RT-PCR) GeneHunter (Differential Display) Competitive PCR Real Time Primer Sets RealTime Probe & Primer Database Ambion (Real Time PCR basics)

TRI REAGENT STEP

(1) centrifuge samples for 10 min at 10C at 1100 RPMI (for adherent cells keep the 24 well plate and add tri reagent to the wells after you have taken off the supernatent. Use this trireagent for step 6)

(2) remove supernatant (throw down sink).

(3) Resuspend pellet with 1 ml PBS (using 1 ml pipette) and transfer into 1.5 ml tubes (the more rounded tubes in RNA cabinet)

(4) centrifuge in small centrifuge (on work bench) (about 30 sec)

(5) remove PBS using vacuum (turn it on in room. Get asperiting pipette (from 2nd drawer and use regulat pipitte tips at end)

(6) Using Bulb pipette, add 1 ml Tri reagent (Sigma, from 3rd shelf) into 1.5 tubes using transfer bulb pipette (2nd drawer). (make sure pellet is resuspended)

(7) let sit for about 10 min

(8) place at -80C (freezer 2nd shelf)

RNA EXTRACTION STEP

(1) Set up Materials [RNA room: 1000, 200, 10 ul pipettes, all specially dept. chlorophorm and isopropynol) (figure how much will need and transfer into 15 ml tube (ex. 6 samples use about 2 ml) (Everything for RNA work should be kept together. This includes tips, racks, pipettes, etc). Clean the work area (something like “conflict” which is a little less aggressive than bleach, then “RNaseZAP” which should be left on surface for 10 sec) Wear gloves. Place 1 special PCR water (sigma) in warming plate and set at low)

(2) Add 200 ul of chloroform to each sample using 1000 tips (otherwise you max out). (Transfer the amount of chloroform need from stock jar to a 10 ml tube rather than take from the stock jar) (ex. If 3 samples take abut 1 ml)

(3) Vortex well until see strawberry like shake color.

(4) Allow samples to sit for 2-15 min at rm temp until see clear layer at top

(5) Prepare new set of 1.5 ml vials and label according to samples. Place samples in 2nd plastic holder and use this to transfer them to cold room

(6) Centrifuge samples 15 min at 12000 rpms at 4C (cold room)

(7) Add 500 ul of isopropanol into new 1.5 ml vials (again dispense from bottle to 10 ml tube 1st) (e.g, 2ml for 3 samples)

Precipitate the RNA

(8) Transfer top clear layer (RNA layer) (using 200 ul pipette) (be careful not to get any pink layer) of sample tubes into isopropanol tubes. Vortex each tube after addition. (dispose of remaining pink layer in special waste container under hood)

(9) Place at rm temp for 5-10 min (or -20C for 1 hr or at -70 overnight)

(10) Centrifuge 12000 for 15 min in cold room

(11) Look for pellet. Carefully remove most of supernatant (using vacuum with special glass tip and regular 200 ul tips. Add 1m of 75% ETOH (kept at -20). Do not disturb the pellet

(to prepare 75% ETOH. .75(100)=75 ml of 100 proof + 25 ml H2O (sigma)

(12) Centrifuge again 12,000 for 5 min at 4C (cold room) (meanwhile prewarm water, see step 15)

(13) Remove all of the ETOH, using vacuum “RNA suckers” (non-yrogenic, RNAse/DNase free 200 ul tips, kept in RNA cabinet; orange box labeled “RNA suckers”; organge box labeled “RNA suckers”) (and when you get close to the pellet use pipetman with tips to move the ETOH away from the pellet)

(14) Allow pellet to dry with cap open for 2-10 min but not so long that pellet drys completely.

(15) Add 20 (40 for larger pellets or 10 ul for smaller ones) ul prewarmed sigma water (comes in a bottle which can be added to vials). Vortex to dissolve the pellet, Pulse spin down using spiner in RNA room and place in 55C warming block for 10 min (or you can freeze for further use) (use timer and after 10 minutes transfer samples to ice)

RNA Quantification

(16) obtain standard reagents (kept at +4C) and 2 new 15 ml tubes (from RNA cabinet) and 1 24 well plate + Sigma Water Bottle (from RNA cabinet), ladder, black plate from RNA cabinet, RiboGreen RNA quantification kit (white top)

(17) Prepare 1x TE Buffer using TE calculator program (<my docs><Jim><TE calculator>)

Ex. stock solution will be 20x so need to dilute 20 times, so you can add 4.9 ml Sigma water and 0.1 ml 20 X TE in our case but this will vary on # of samples) (just insert # of sample in TE calculator on Disk which you can print out)

(To prepare new standards: (ex. 5 ml) We have 20x TE buffer in kit and want 1x.

1ul (TE):20ul (sigma water) 2ul:40 ul 3ul:60ul 4ul:80ul 5 ul: 100ul so 50 ul for every 1000 ul sigma water or more precisely 50 ul for 950 ul water So 250 ul TE for 4750 ul sigma water

Obtain 4 new 2 ml (round btm tubes) and replace set according to directions on container. 1st add appropriate amounts TE with glass pipette. Then add appropriate amount from tube (ribosomal RNA standard marked with *) with pipette.

(18) Prepare Dye in 2nd tube by adding amount of dye (ribosomal quantification; blue cap) according to TE calcualtor program.

Ex. add 1.6 ml 1x TE + 8 ul 200x dye stock)

(19) Take samples from warmer and put straight onto ice. Prepare 1:500 dilution of RNA samples in 24 well plate. Place 2 ul RNA in well and add 1ml TAE Buffer (1X) to give a 1:500 dilution [2:1000ul=1:500]. Shake the plate with hand. (you do not need to change glass pipette if not touching sides)

Sample #1 1:500
Sample #2 1:500
Sample #3, etc

(20) Get black well plate (in RNA cabinet). Place black plate up at an angel. Add the following to the appropriate wells.

A1: (blank) 100 ul 1x TE only B1-E1: 100 ul standards

A2: 100 ul sample dilution 1, B2: 100 ul sample #2…. Skip column 3

A4: 10 ul dilution sample 1 + 90 ul 1X TE: B4: 10 ul dil sample 2 + 90 ul 1x TE (This will give a dilution factor of 1:5000 because each of the samples are at a 1:500 dilution and by adding 90 ul TE to 10 ul of the sample, you are diluting by a factor of 10. So the 500 would become 5000.

1 2 3 4 5

(Blank Well) 100 ul 1X TE

100 ul dilution sampel1: 1:500

10 ul Dilution sample #1 (from 24 well plate) + 90 ul 1x TE 1:5000

100 ul std #1 40 ng/ml

100 ul dilution sampel2: 1:500

10 ul Dilution sample #2 + 90 ul 1x TE 1:5000

100 ul std #2 200 ng/ml

100 ul std #3 1000 ng/ml

100 ul std #4 2000 ng/ml

Problem: We want 5 ml of 1XTAE buffer. How do we prepare from stock of 20x TAE. Answer: 1 +

19 ml H2O is 1X. But we want only 5 ml. So divide everything by 4. 1/4=0.25 ml + 4.75 H2O.

(21) Add 100 ul of dye to each of the wells (including the blank) above. Doing this will change the concentrations of each well (the concentrations of the standards will be cut in half and the dilution factors increased 2 fold) as outlined below. (if you do not touch the sides can use single pipette tip)

1 2 3 4 5

1:1000

1:10000

20 ng/ml

1:1000

1:10000

100 ng/ml

500 ng/ml

1000 ng/ml

(22) Sign up for fmax. Turn machin on (preferably 5 min before use). Open machin using icon on right top <M>. Place plate in fmax. Close dorr <D> icon to right and top. Open soft max program on desktop. <file “cathy” <Ribog10.PDA>

<template> Enter concentrations of standards under “sample” and “dilution factors” for samples if necessary.

<Read>

Print out copy for records (select only page you need. ex. pg. 3-3)

exit program

Reading will be in ng/ml so need to convert to mg/ml by moving the decimal 6 places to the left (eg., 10-9 ng becomes 10-6 ug). The reading will enable you to determine how much of your RNA sample you will need to get 3 ug RNA. Dots next to #s mean dilution is wrong.

Note: if there are (.) dots next to your numbers, you will need to choose other numbers because this means your #s are out of range (too concentrated). If all #s like this will need to dilute. Print page. Close fmax using icon top right.

(23) Use the results to input into excell program that will tell you how much RNA from your sample to add to get 3 ug of RNA. (if 3 samples, input 4 on the excel program)

Sample RNA (ug/ul ) RNA to Add [ul req. for 3ug] Water to Add

1. sample	reading says 0.51	5.9 (must add this because 3 ug RNA/0.60=5)	4.1
2.	.
3.

DNAse Treatment (optional)

(1) label 0.5 PCR tubes according to # of samples

(2) Go to excell program (tells you how much RNA to add) Print out and hang up on hood

Sample	ug/ml	8ug (want to use of RNA for DNAse	H2O	DNase	Buffer	Final
1. Med		ex. 6.3 (add 6.3 ul sample to get 8 ug/ml of RNA)
2. LPS
3. LPS/THC

(3) add appropriate amounts to PCR tubes.

(4) spin down. Will get 2 layers 1 clear, 1 milky. Transfer clear layer (RNA w/out DNAse into PCR tubes (green top)

(5) leave in water bath for 45 min at 45C.

(4) Recalculate/quantitate amount of RNA as above

RT-PCR

(1) clean hood with conflict 1st, then RNase. Obtain tips, bucket with ice, small ice rack (in RNA cabinet to hold tubes),Lable 0.2 ml PCR tubes and place in the tube ice tray.

(2) Use Excel spreadsheet from above to determine how much of the rest of reagents will need to add for master mix (in addition to the RNA and water above). You will need to add your # samples + one extra. Your total volume for the MM per sample is 10 ul composed of the reagents below.

(3) place your samples form the freezer in ice bucket.

(4) Get materials from PCR bucket (-20C) and dNTP from RT-PCR bucket.

(5) Lable an appropriate # 0.2 ml PCR tubes and place in the tube ice tray. Also get a small sigma water tube and 1.5 steril tube for the master mix.

(6) Add appropriate amount of H2O (calculation sheet next to “sample”) and sample RNA into PCR tube(s). Ex. for 2.90 ug/ul, add 1 ul RNA + 9 ul water) Pulse spin to get H2O to bottom.

(7) Place samples in thermocycler and perform 95 cycle (5 min)

<turn on><files><load><95><enter><T=95.0 5:00 2. hold 4.0><close><exit><exit><start>

(8) Prepare Master Mix according to sheet (use total mix calculations).

EX. For 6 samples you would get the following

Total Mix

AMV (always add last), just before add MM to sample)	1.5	9
RT buffer	4	24
dNTP	2	12
RNasin (RNA inhibitor)	0.5	3
Oligo (this is primer)	2	12
Total	10

(9) take PCR tubes with samples out of cycler and place on ice tray under hood.

(10) Add 10 ul of MM into each sample.

(11) Pulse spin tubes. Place tubes in thermocycler for 45 min (program called “CNRT” (45 min at 42C. Then 5 min at 95C (to stop rnct), then 4C hold). To start program press <exit><exit><start>(At this point you can stop and store your products at +4 up to several weeks or can continue)

PCR

(1) take out PCR excell sheet (prepare for # samples + 1 extra)

(2) Mix forward and reverse primers into 1 tube. (just tranfser 1 tube to the next and relable the tube as “Mix” and place primer name on top)

3) Label 0.2 ml PCR tubes and place in ice tray. Use different colored tubes for different primers. (bring bags with tubes out)., also get 2 tubes (1.5 for PCR) and another sigma water tube.

(4) Get the PCR materials (most of which are in PCR bucket) but dNTP is in the RT-PCR bucket.

(5) Prepare Master Mix below except that do not Add Ta1 yet. Again, you can prepare a program in Excel (can be the same sheet as above) to figure out how much of each reagent you will need for your mix. You can add everything except the primers to all of the tubes since everything else will be the same (just use same tip and dont touch)

Ex. The total volume for each sample here is 23 and we have 6 samples. so:

for 6 samples (varies depending on rnct)

Water (Sigma)	16.75	100.5
10x PCR buffer	2.5	15
Primers Mixture 1	1	6
dNTP	2.5	15
Taq (But do not add this yet!)	0.25	1.5
Total	23	138

(6) Add 2 ul of your RT cDNA (from the thermocycler) to each new sample tube you prepared above. Also add 2 ul water instead of RNA to a separate tube.

(7) Now add your Taq to the to the master mix

(5) Add 23 ul of the master mix to sample tubes. Also add to neg control (the one with water instead of RNA) pulse spin

(6) Place tubes in thermocycler and run protocol (–turn on, place tubes in wells, lock in place, –file enter–load enter -CM60 enter) step 5: (6 to (28) varies depending on sample run. <exit><exit><start>)

PREPARE AGAROSE GEL

(1) obtain agarose from chemical shelf and TE buffer 1x. Get agarose beaker and top

(2) set up gel place by placing metal plates at ends.

(3) make 100 ml of 2% agarose. So 2 g agarose + 100 ml TE buffer (prepare TE buffer by adding 490 nonopure water _ 10 ml 50x TE)

(4) take the beaker to kellys lab and place in microwave on paper towel. heat 1 min (60 sec +start). Repeat 2 times and shake bottle in between heatings. Agarose should be fully dissolved

(5) cool beaker in ice bucket in sink filled with water (just hold beaker in bucket. don’t create air bubbles)

(6) Add 5 ul ethidium bromide using special pipette next to flask

(7) pour contents of beaker into gel plate (keep distance since carcinogenic!) If any air bubbles are created use pipette tip to move aside

(8) place 2 combs; 1 to left forward and one middle.

(9) wash flask immdiately with hot water first then distilled water. Turn upside down on paper in room

PCR THERMOCYCLER

Turn on cycler in back

Opening Files

(1) <Files><enter>

(2) <load><JRGRAD><enter>

Saving Files

(1) <exit>

(2)<save><yes>

Setting the Gradient (to find the columns)

(1) <options><enter>

(2) <gradient><enter> (ex. T=50, G=5.0)(this means range 55-50-45) (each column will be diff temp; write down the column temps) (we use small blue tubes so small holes)

(3) Set your gradient temp and cycles

LID=(lid temp)

1) Initial denature T=92.0 3.00

2) Denature T=94.0 15

3)Annealing T=62 (hit the <opt> to condense step 3

4) Extension T=72

5) Go To 2 Rep 39 (so this is really 40 cycles because already ran once)

6) Last extension T=72

7) hold 4.0C (when cycle completes move samples to refrig)

Load the Gel

(1) get 100 bp ladder from fezzer part. (will laod 5 ul ladder)

(2) Bring Big well plate forward (add 1x TE to plate if necessary to cover gel)

(3) Take well plate out of small plate, bring up in air and remove metal plates. Put the Gel plate under TAE buffer in large plate.

(4) remove combs. Get 96 well culture plate (use to laod samples) cut towel for place on bench and work on this.

(5) Load ladder (usually put ladder in center. load 5 ul) Usually leave end wells open. If samples are not colored will have to use Dye (1:4; since 4X dye, use 4 ul dye for 16 ul sample). Mix the samples with dye in 96 well plate. If samples are colored can load directly 16 ul into wells. (use gel loading tips)

Load samples form left to right. (ex. green tubes (p40) left and p35 right)

(6) run gel for 50 min at 100 volts or 45 min at 110 (select V for voltage and timer (45 min) then hit little runner)

Taking Picture of Gel

(1) connect leads to camera (power button on camera)

(2) turn camera on

(3) <kodack ID> icon (Kodack 1.D.3.5)

(4) <capture DC290> (screen appears never change paremters. should say default. sample type: SYBRGREEN (gives better pciture) sample size: exposure time (3.5 sec)

(5) turn light on only for a few seconds while taking picutre

(6) <take preview> shut off light when picture comes on

(7) turn light on momentarily , Then <export image> (after a few seconds will hear click, then turn light off)

(8) save image

(9) <close>

(10) <rotate> picture b4 saving it

(11) <save as>

(12) can also take regular picture as back up.

(13) wash gel plate with distilled water.

Measuring Intensity with Kodak Machine

(1) <ROI “Region of interest”> icon on left <2nd box (square)> highlight 1st band (make box)

(2) <make a grid> Ex. <colum-4> row=1 fro 3 bands. Hit arrow key so you can separate boxes away from each other

(3) read the intensity <show> <ROI> <analyze data>

(4) export data <file> <export data> <ROI> <as displayed> <put in folder>

(5) bring up saved program in excell <close program> <excel> <file> <open> <test files> since saved as text. text import wizard comes up <next><next> <finish>

(6) only interested in mean intensity so format the box <format><cell><#> 2 decimals. print out

Resuspending Primers

(1) Prepare 1X TE buffer. Make from 20X TE buffer RNase free small bottle at 4C. (Ex. you need 1 ml. so 950 ul sigma water and 50 ul TE buffer) (Always transfer sigma water from the bottle to tube first)

(2) Add TE Buffer into vials containing primers. The Quality Assurance Document will tell you “nmol” This is the amount of TE to add.(ex. IL12p35F=52.3 ul).

(3) vortex and spin down

(4) from each vial take 10 ul and add 190 water. This gives a 1:20 dilution. [before use is 1 mM; we want 50 uM] Dispesne these amounts into PCR tubes

(5) store at -80C (where the samples are kept) except that for 2 of the tubes (f and r) which you should combine (just transfer from one tube to the other) and relable this tube “mix” and date. This is working stock to be kept in freezer part of lab refrigerator.