Bcr-abl1 monitoring test, Reaction set up- | Homework Helpers

Reaction set up- BCR/ABLtranscripts

You will assemble a ‘master mix’ with sufficient volume to perform 2 x 20?L (repeat) reactions for each transcript level quantification. Remember to change pipette tips after pipetting each solution (to avoid cross-reagent contamination).

Don't use plagiarized sources. Get Your Custom Essay on
Bcr-abl1 monitoring test, Reaction set up- | Homework Helpers
Get an essay WRITTEN FOR YOU, Plagiarism free, and by an EXPERT!
Order Essay

• Pipette 16.8 ?L of the BCR/ABL primer mix into a 1.5mL tube
• Pipette 4.2 ?L of the K562 cDNA into the tube
• Pipette21 ?L 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 20?L (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 ?L of the GUSBprimer mix into a 500?L PCR tube
2. Pipette 4.2 ?L of the K562 cDNA into the tube
3. Pipette21 ?L 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 aIDing 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, aID 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), aID 15?L 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 laIDer 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 ?L of the DNA laIDer
2. Into the second well load 20 ?L of the GUSB qPCR reaction
3. Into the third well load20 ?L 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 ?L 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 10?L 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.

Calculate your paper price
Pages (550 words)
Approximate price: -

Why Choose Us

Top quality papers

We always make sure that writers follow all your instructions precisely. You can choose your academic level: high school, college/university or professional, and we will assign a writer who has a respective degree.

Professional academic writers

We have hired a team of professional writers experienced in academic and business writing. Most of them are native speakers and PhD holders able to take care of any assignment you need help with.

Free revisions

If you feel that we missed something, send the order for a free revision. You will have 10 days to send the order for revision after you receive the final paper. You can either do it on your own after signing in to your personal account or by contacting our support.

On-time delivery

All papers are always delivered on time. In case we need more time to master your paper, we may contact you regarding the deadline extension. In case you cannot provide us with more time, a 100% refund is guaranteed.

Original & confidential

We use several checkers to make sure that all papers you receive are plagiarism-free. Our editors carefully go through all in-text citations. We also promise full confidentiality in all our services.

24/7 Customer Support

Our support agents are available 24 hours a day 7 days a week and committed to providing you with the best customer experience. Get in touch whenever you need any assistance.

Try it now!

Calculate the price of your order

Total price:
$0.00

How it works?

Follow these simple steps to get your paper done

Place your order

Fill in the order form and provide all details of your assignment.

Proceed with the payment

Choose the payment system that suits you most.

Receive the final file

Once your paper is ready, we will email it to you.

Our Services

No need to work on your paper at night. Sleep tight, we will cover your back. We offer all kinds of writing services.

Essays

Essay Writing Service

You are welcome to choose your academic level and the type of your paper. Our academic experts will gladly help you with essays, case studies, research papers and other assignments.

Admissions

Admission help & business writing

You can be positive that we will be here 24/7 to help you get accepted to the Master’s program at the TOP-universities or help you get a well-paid position.

Reviews

Editing your paper

Our academic writers and editors will help you submit a well-structured and organized paper just on time. We will ensure that your final paper is of the highest quality and absolutely free of mistakes.

Reviews

Revising your paper

Our academic writers and editors will help you with unlimited number of revisions in case you need any customization of your academic papers