Unit 1: week 5
Yeast Mating “The Effect” Part IIIb
Blackboard Assignment due by Monday 9:59 PM
Reading: Last week you took great care isolating RNA. This week we begin the
Comparative RT-PCR procedure. Make sure you understand what reverse transcription
and PCR are and what the RT-PCR reaction will do.
**This week, in the interest of time I have performed steps 1-10 with the ‘best’ induced
and non-induced samples the lab isolated last week. (‘Best’ means best balance between
highest amount and least protein contamination). Be sure that you understand what was
done. While characterizing your mutants you would need to be able to perform these
steps yourself. And, you are responsible for the theoretical material behind the steps that I
perform.
____________________________________________________________
You are still working with RNA. Before you begin be sure to wipe down your lab bench
with RNAse removing detergent and only handle samples while wearing gloves.
Reverse Transcription and adding Tag10 and Tag50 tags
1) Get an ice bucket, retrieve your RNA samples from the freezer and allow them to
thaw on ice.
2) For the next steps you need 5ug of RNA.
DLY1 Non-induced--> 5ug= ____ul
DLY1 induced --> 5ug= ___ul
(Use the concentration you determined last week for this calculation)
*If the volume calculated is greater than 13.5ul do not proceed—talk to me.
If you are prepared, part of the group can prepare the RT-Primer Mix while the other part
prepares the Master Mix
Prepare RT:Primer Mix
We will be tagging the DLY1 non induced RNA’s with Tag10 and
DLY1 induced RNA’s with Tag50.
(Using this labeling—If we examine the expression of geneX by RT-PCR which sample
will always have a gene X band with a higher molecular weight? If it has a higher
molecular weight band where will it appear on the agarose gel (closer to the wells or
closer to the bottom of the gel?
3) On ice, mix 5ug DLY1 non-induced RNA with 1ul Tag10 RT primer (side bench) in a
500ul PCR tube (small RNase-free microfuge tube). Make sure tube is labeled
NOTE: remember you have to fill in the # of ul required to mix 5ug
4) In a separate tube, on ice, mix 5ug DLY1 induced RNA with 1ul Tag50 RT primer
(side bench) in a PCR tube. Make sure tube is labeled
Master Mix Preparation:
5) On ice, prepare a master mix for each RNA sample. Each master mix contains
a) 2 ul 10X RT buffer
b) 2 ul RT dNTP mix
c) 0.5 – 13 ul nuclease free water to bring the final volume of the RT reaction to 19
ul (including the volume of the RNA:RT Primer Mix that will be added later.)
Ex. If you use 10 L of RNA + 1 L primer +2 L buffer + 2 L dNTPs = 15 l,
so you need 19-15 = 4 L of water.
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The RT Reaction:
The Thermal Cycler has been programmed for the following steps
Step 1: 5 min @ 72C
Step 2: 5 min @ 49C
Step 3: 2 hr @ 49C
Step 4: 4 C hold
6) Place the RT:Primer Mixtures into thermal cycler. Let them proceed through Step 1.
This step denatures the RNA. (RNA is single stranded what could need to be denatured?)
7) When the thermal cycler reaches Step 2, place the Master Mix in the machine.
This step allows the RT-Primer mix and master mix to equilibrate at the optimal
temperature for reverse transcription with these primers.
8) When the thermal cycler reaches Step 3, pipet the correct amount of master mix into
each corresponding RT:Primer tube.
9) Then, immediately, add 1ul of reverse-transcriptase to each tube and complete Step3.
10) When the machine cools to 4C remove the samples and place them on ice (or store
them in the freezer for later processing)
*****This week you will start here: Using one Tag10 and one Tag50 RT reaction to be
shared by everyone in the lab section.
Preparing the cDNA for PCR
Now you have complementary DNA (cDNA) that is tagged with either Tag10 or with
Tag50. Be able to describe the number of different cDNAs found in each of these
samples in relative terms. What was the RT copying and so what do you have in each of
these cDNA ‘pools’?
The cDNA is not quite ready for PCR yet. Currently it sits in a buffer full of unused
primers and nucleotides that didn’t get use in the RT reaction. In the PCR reaction we are
about to perform, one of the PCR primers binds to a region found on each ‘Tag’ primer.
Leftover/unused Tag primers also contain that region and would, therefore act as a ‘sink’
for PCR primers and alter the number of PCR primers available for interaction with the
true template—the cDNA. Therefore we need to remove all free nucleotides or
nucleotides chains that are shorter than ~75bases.
Last time we wanted to remove unwanted DNA we just added DNase to digest it. That
approach can’t be used this time. (Why not? What has changed about our sample)
Fortunately the primers and unincorporated nucleotides are much smaller than our
cDNAs. We can remove them by preferential binding and size separation. This is done by
using a column called ‘NucAway’.
Prepare NucAway Columns:
11) Tap the column to settle the dry gel in the bottom of the spin column.
12) Add 650 L of water to hydrate the column. Cap, vortex, tap out air bubbles, and
hydrate at room temperature for 5-15 min (15 min is better).
13) Just before you’re ready to use the column, remove the bottom cap, place the column
in a 2 mL tube and centrifuge at 750 x g (~30%) for 2 min to remove excess
interstitial fluid. KEEP TRACK OF THE ORIENTATION OF THE COLUMN IN THE
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ROTOR- mark the outer edge with a sharpie. (also place mark on it so you know which is
yours and what sample will go in it in the next step)
cDNA NucAway:
14) Label a 1.5ml tube for each sample.
15) Discard the 2ml tube, put the column into a 1.5 mL tube, remove the upper cap from
the column, and apply your cDNA sample directly to the center of the gel bed at the
top of the column, without disturbing the gel surface or touching the sides of the
column with the pipet or reaction mixture.
16) Put the column in the rotor, maintaining the same orientation as before by placing
the sharpie mark toward the outer edge.
17) Centrifuge at 750 x g (~30%) for 2 min. The tagged cDNA will run through to the
collection tube. Discard the spin column.
cDNA Quantitation:
18) Quantitate the cDNA via spectrophotometry at OD260 (using the disposable ultramicrocuvettes, same as last week except use 3ul of sample in total of 200ul) Do not
forget to use correct new your dilution factor accordingly in your concentration
calculations
DLY1 non-induced Tag10 cDNA OD260=
DLY1 induced Tag 50 cDNA OD260=
ug/ml=
ug/ml=
19) Look at the cDNA concentrations from #18. Dilute the more concentrated one with
Nuclease free water so that it is the same concentration as the less concentrated one
cDNA quantitation is critical to the ‘comparative’ part of Comparative RT-PCR. In the
PCR section we will be combining cDNA samples—always using one Tag10 labeled and
one Tag 50 labeled sample. When the PCR proceeds there will be a certain amount of
primer and the cDNAs from the two ‘pools’ compete to bind those primers. Remember
this is not a competition where one set of cDNA’s can have more muscle or run faster. It
is a competition where random interactions are solidified by complementary binding
(everything mixes together and if the ‘right’ nucleotide partners are found the two strands
will stick together).
Think about why concentration matters. What would happen if primers were mixed in a
tube with 1ug cDNA from sample B and 10ug of sample C? 1ug sample B and 2ug of
sample C? All of a sudden you would not be measuring the amount of differential gene
expression— just the fact that you added more of one sample than the other.
We want to determine the relative amount of a gene’s cDNA present due to different
growth conditions. Not amounts due to adding 10x more of one sample than of the other
so quantitation is very important.
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PCR Part one: setting up outer primers reactions for nested PCR
This protocol is new and its development is due months of optimization performed by
Victoria Statler ’03. One of the things that Victoria determined was that, with our
primers, products were only visible if ‘nested’ PCR was done. Nested PCR is a two-stage
process diagrammed below.
Gene-specific Primer
PBS1
PBS2
Binding site
Pool of cDNAs
PCR using a gene
specific primer and one
that binds with PBS1
Many
copies of
target
region
PCR using a gene
specific primer and one
that binds with PBS2
Many, many
copies of
internal
target region
Further analysis
In the first set of reactions the cDNA for our gene of interest is specifically amplified
from the pool (‘standard’ PCR). (Why?/How?). Unfortunately we have found that we still
cannot detect this amount of product. Therefore a second round of ‘nested’ PCR is
performed using primers that are specific to our gene but slightly closer together on that
same DNA piece. This second round of PCR has been enough to allow analysis of the
resulting fragment.
PCR reactions will always be set up in my presence—if designing experiments for the
independent projects you will need to a) set them up during scheduled lab periods (T or
W) or b) make arrangements with me ahead of time.
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The thermal cycler program is as follows (PreProgrammed into PCR machine as “VIC”):
Step 1: 5 min @ 94C denature
Step 2: 30 sec @ 94C
Step 3: 30 sec @ 50C
Step 4: 30 sec @ 72C
(35 cycles of Steps 2-4)
Step 5: 10 min @ 72C
Step 6: 4C hold
Make sure that you have
 Ice bucket
 Thin-walled PCR tubes—handle with gloves
 Your cDNA samples (always kept on ice)
 Your gene specific primer in 2M stock concentration
The following are all from the Amhion Intraspec kit
 Outer Tag Primer (from Ambion Intraspec kit)
 10X PCR Buffer (from Ambion Intraspec kit)
 PCR dNTP Mix (from kit)
 10X Acetamide (from kit)
 Nuclease-free water (from kit)
 Taq polymerase. (keep in freezer until needed, never taken off of ice)
Gene Specific primer choices are:
TUB2, KAR1, STE4, and FUS3
To maximize data from this portion of the lab each lab group should choose a different
gene specific primer. Be sure that you know what the gene products of these gene are and
what they do so that you can form a hypothesis about changes in expression levels.
Prepare Master Mix:
20) On ice, in a ‘regular’ microcentrifuge tube prepare a master mix for four reactions.
Each master mix contains
50.4 L Nuclease-free water (12 L)
10.5 L 10X PCR Buffer (2.5 L)
10.5 L PCR dNTP Mix (2.5 L)
10.5 L 10X Acetamide (2.5 L)
4.2 L Outer Tag PCR Primer (1 L)
4.2 L 2 M Your group’s Gene Specific Primer (1 L)
21) Pipet 21.5 L of the master mix into four thin-walled PCR tubes. (on ice)
22) Add cDNA and Water
To each of the 4 reaction tubes, add the correct amount of cDNA and water according to
the chart below. These are the correct controls for one gene-specific primer. You have
already diluted the cDNA samples so adding equal volumes will add equal ug amounts.
Mark the lids so that you can tell both which reaction it is and which group set it up.
Exact measurements are critical—use the p2 to add the cDNAs or water.
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PCR
Comparative
Tag10 control
Tag50 control
No template control
Nuclease-free H2O
0
0
0
2 L
Tag10 cDNA
1 L
2 L
0
0
Tag50 cDNA
1 L
0
2 L
0
The Reaction:
3) Place the reaction tubes in the thermal cycler. Let them get ~ 30 sec into Step 1 @
94C.
4) After 30 sec push ‘pause’, open the lid to the thermal cycler and add, using the p2,
0.5 L of Taq to each reaction tube.
5) Allow the PCR to occur according to the program outlined above.
The program takes approximately 2.5hrs and ends with storage at 4°C for 24 hours. For
this session I will move the tubes when the reactions are done. When performing this type
of experiment with your mutants you will have to come back and move your samples.
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