Reaction set up- BCR/ABLtranscripts
You will assemble a ‘master mix' with sufficient volume to perform 2 x 20μL (repeat) reactions for each transcript level quantification. Remember to change pipette tips after pipetting each solution (to avoid cross-reagent contamination).
• Pipette 16.8 μL of the BCR/ABL primer mix into a 1.5mL tube
• Pipette 4.2 μL of the K562 cDNA into the tube
• Pipette21 μL of the x2 BioRad iTaq qPCR mixinto the tube
• Gently pipette the mixture up and down 3-4 times to mix, then flash spin in a centrifuge
• Bring your reactions, on ice, to the Thermal cycler. You will be directed where to pipetteyour master mix into 2 wells of a 48 well qPCR plate (make a note of your well numbers!)
• Once all samples are loaded, we will then run the qPCR and use the CFX manager software package to observe the ‘real-time' data and extract the final qPCR values.
Reaction set up- GUSBtranscripts
Each studentwill assemble a ‘master mix' with sufficient volume to perform 2 x 20μL (repeat) reactions for each transcript level quantification. Remember to change pipette tips after pipetting each solution (to avoid cross-reagent contamination).
1. Pipette 16.8 μL of the GUSBprimer mix into a 500μL PCR tube
2. Pipette 4.2 μL of the K562 cDNA into the tube
3. Pipette21 μL of the x2 BioRad iTaq qPCR mixinto the tube
4. Gently pipette the mixture up and down 3-4 times to mix, then and flash spin in a centrifuge
5. Bring your reactions, on ice, to the Thermal cycler. You will be directed where to pipetteyour master mix into 2 wells of a 48 well qPCR plate (make a note of your well numbers!)
6. Once all samples are loaded, we will then run the qPCR and use the CFX manager software package to observe the ‘real-time' data and extract the final qPCR values
Preparing an Agarose gel and running buffer
1) Pour a 50 mL aliquot of 10X Tris acetate electrophoresis buffer (TAE) stock solution into a 1 litre-measuring cylinder and make up to 1L by adding 950 mL of deionized water. This will create a 1L stock of 0.5X TAE buffer.
2) Assemble a gel-casting mold by attaching the red rubber stoppers to the two open sides of a casting tray. Place the completed assembly on a flat surface. A good idea is then to test the casting try for leaks. Do this pouring a small aliquot of 0.5X TAE buffer into the mold and observing for ‘leaks'.
3) Weigh out 1.2g of agarose into clean 250 mL bottle, add 150 mL of your 0.5X TAE buffer and gently ‘swirl' to mix the agarose/TAE slurry.
4) Heat the slurry in a microwave until the agarose fully dissolves. Be careful, not to let the slurry boil over. About 4 minutes on medium-high power should be sufficient.
5) When the solution has cooled sufficiently, (to approximately 55°C), add 15μL of x10,000 SafeView DNA stain dye to the gel mixture, gently swirl to mix and pour the gel solution into the gel-casting tray. If needed, remove any bubbles that may have formed with a clean pipette tip.
6) Place 2 combs into gel: one into the slots at the top of the gel mold and the second ‘half way down' the gel, then leave the agarose gel to ‘set'. Take care not to disturb the gel until it has set. The gel is set when it appears milky white and translucent.
7) Once set, carefully remove the rubber stoppers from both ends of the mold
8) Transfer the whole gel try into an electrophoresis tank and fill the tank to just under the ‘max fill level' with 0.5X TAE buffer.
9) Remove the comb from the gel, taking care not to break the wells. A demonstrator will help you to do this if required.
10) Your gel is now ready to be loaded.
Preparing your qPCR reactions for gel loading
You have been supplied with the following reagents:
3 x 1.5mL tubes -
Tube D contains a x6 concentrated red coloured solution of DNA loading dye
Tube L contains a DNA ladder solution
Tube SV containing SafeView DNA stain solution
1 x 50mL aliquot of 10x TAE buffer
Loading the gel
Each student should load 3 wells of the ‘group gel'.
1. In the first well load 5 μL of the DNA ladder
2. Into the second well load 20 μL of the GUSB qPCR reaction
3. Into the third well load20 μL of the BCR/ABL qPCR reaction
Use a p10 or p20 pipette to load the samples into the slots of the wells. Remember to use a fresh pipette tip for each well loading. Note you can use 10 μL tips on the p20 pipettes. These tips have ‘finer' points making it easier to accurately load your samples into the wells. Do not try to load all the sample in one go, pipette the sample into the wells in aliquots, 2 x 10μL for example. Demonstrators will be available to help you load your gels if required. Make sure you note which wells contain which samples!
Discussion
This should be approximately 800 words (excluding any figures, tables and legends)
The discussion MUST be correctly referenced with relevant literature from peer reviewed scientific sources. The discussion should have 3 sections.
• Section 1: Discuss the final results you obtained for each stage of the results. Clearly explain the main findings from each section and how one results section leads on to the next. Highlight any issues that may have affected the accuracy/precision of dour results and/or progression from one stage to the next stage. This section should be a logical progression of the ‘flow' of the whole experiment/practical from start to finish.
• Section 2: You should then come to a final conclusion discussion point as to the success or otherwise of the entire experiment(s). What conclusions can you draw from the data and the final result?
• Section 3: Finally you should discuss what would you and/or other researches could/should do next with your findings. Discuss what's been previously beeb published in the literature about the topic. State what the key impact of your results in relation to these previous findings is. Why should other people care about this ‘result' and what could you or they do to further it/act upon it.