Teachers notes
Splicing Lab
Purpose: To understand that RNA is genetic material and also an enzyme. To understand
that genes are spliced together at the RNA level.
Background:
RNA is genetic information, it serves as a message to make proteins. It is important that
the RNA message is put together correctly in order to make the right protein. In order to
do this, RNA messages are cut and pasted together, a process known as splicing.
Splicing is a way to cut and paste genes together. Why do genes need to be spliced?
Splicing removes part of genes that do not code for protein. It also adds variety to genes,
since they can be spliced (put) together in different ways. The parts of genes that code
for proteins are called exons, the part of genes that do not code for proteins and are cut
out are called introns. Introns are usually spliced (or cut) out of a gene.
How does splicing occur? Splicing occurs with the help of enzymes. Enzymes are
molecules that speed up a reaction. Some enzymes are proteins, but RNA can also be an
enzyme. An RNA that is an enzyme is a ribozyme. Some ribozymes are capable of splicing.
Ribozymes can cut out introns and splice their own gene. The ribozyme is in the RNA
message itself, located in the intron.
PRE-LAB questions – Do prelab questions together as a class
What is an enzyme?
A molecule that speeds up a reaction (by lowering the activation energy)
Some enzymes are proteins, but enzymes can also be ___RNA___.
Splicing is a way to __Cut__ and __Paste___ genes together
Materials
DNA of Ribozyme
37 degree incubator
Agarose
Centrifuge tubes
Transcription kit (promega)
P2075
UV Light source
Electrophoresis gel box
Electrophoresis Power supply
Sybr gold (invitrogen)
S11494
Things you may need to buy:
Transcription kit can be purchased from promega, item # P2075
Sybr gold can be purchased from invitrogen, item # S11494
DNA of Ribozyme can be donated from the Luptak lab (small aliquot, enough for 10uL
reactions)
Methods for teacher:
1. Transcription
Transcription can be set up by teacher or resident scientist, the reagents
are expensive. If students are trust worthy, you can ask students to do it.
To set up Transcription for 10uL reaction (using kit listed above)
Reagent
Volume
Transcription 5x buffer
5uL
DTT 100mM
1uL
RNTP mix
1.5uL
DNA
1uL
Phage RNA Polymerase
.5uL
H20
1uL
*NOTE – You can have students add in the 1uL of H20, but tell them they
are adding the Polymerase enzyme.
2. Agarose gel
Make 1% gel.
A. Weight out 1 gram of agarose into 50mL of TBE buffer.
B. Microwave for 1 minute until the agarose has dissolved.
C. Pour agarose into a gel cast. Make sure a comb is inserted
D. Add 1uL of sybr gold to each groups final product before loading
the gel
E. Load and run gel (I suggest 200V for 15 minutes)
When students are ready to load their sample, you can load for them
or have them load it.
I suggest doing a full agarose lab previous to this lab. If they do an
agarose lab/activity the week before, they can make the gels for the next
week and keep them refrigerated.
Methods for students:
PART A – Transcribe DNA to RNA
Each station has a tube labeled DNA and P (for Polymerase).
1. Add 1uL of Polymerase from tube P to the tube labeled DNA.
PART B - Incubation
1. After 1uL from tube T is added to the tube DNA, place the DNA tube in a 37°C
incubator for a certain amount of time depending on what group you are in. Label your
tube with what group you are in.
Group 1- 5 minutes
Group 2 – 10 minutes
Group 3 – 15 minutes
Group 4 – 20 minutes
PART C – Visualize splicing
1. After your time is up hand your teacher or resident scientist your tube. Make sure it is
labeled with your group number.
2. The tube will be run on a gel. Draw an image of the gel after the gel is done running.
Indicate where your group is on the gel.
*Your teacher will run a control in the first lane. The control has not been
incubated at all. How many bands should you see in the control #_1__?
Questions
1. A ribozyme is an example of an_______enzyme_______________.
2. What is an intron? What is an exon?
Intron – Does not code for protein, is cut out
Exon – Codes for protein, spliced together
3.
After RNA is made and spliced, what does it code for?
Protein
4. What is the function of the ribozyme in splicing?
To cut out exons, to prepare RNA to be made into protein
5. On a gel, a single piece RNA that does not splice will contain #_1__ bands, while an
RNA that splices once will contain #__2 or more__ bands.