Can someone please help me with one and two Pre-Lab 2.5 Due Tuesday by 10am Pint
ID: 205768 • Letter: C
Question
Can someone please help me with one and two Pre-Lab 2.5 Due Tuesday by 10am Pints 3 Submitting on paperAvailable until May 4 at 11:59pm 1. Perform the necessary calculations for each sample that you wil load on the gel in part I. 2. When designing primers you should have 3 G's or C' in the last 5 bases on the 3 end, but no more than 3. Explain why Lab S Today you will run a gel of your digests from last week and analyze the results Alfter running the gels we wil determine which segments of the lambda genome wr have coned While the gels run you will design PCR primers to mutate your assigned gene Safety notes: Ethidum bromide is a potent mutagen Wear gloves when handling containers containing ethidium bromide, when handling gels that have been stained in ethidium bromide, and when working with the computer attached to the gel-doc system Protocol: L Loading and running the gel 1 Prepare your samples (the 3 digest reactions from last week) for electrophoresis. The final volume for each sample that you load should be 20 For each sample you should load 3 l of digested DNA Each sample needs loading dye (supplied as Sk, you want it to be 1x). Use TE to balance the volume for each sample 2. Load your samples and a Mend standard Be wre to record which sanglesgo Run the gel at 120V for at least 1 hour 4. Stain the gel in ethidium bromide on the shaker table for 30 minutes 5. Take a photo of your gel. Determine which segments of the lambda genome were cloned into each of your white colonies. Report this information to Rachel Il Designing primers for PCR mutagenesis Use this website to des, your otide pr mers ttp://www.ncbi.nim.nih gou/tool/primer-blast Use these guidelines to design your inside primers Primer length: 18-24 bp GC content 4060% GC dlamp: in the last 5 bases on the 3 end you should have 3GsorC, but no more than 3Explanation / Answer
1. Total volume= 20 microliters
DNA added is 3 microliters of PCR digest
Stock of loading dye is 5X. To obtain 1X, dilute this stock 1:5. For 20 microliters final volume, add four microliters.
TE buffer= 20-(4+3) = 13 microliters.
Final Sample to be loaded on gel: 4 microliters of dye+ 3 microliters of PCR digest + 13 microliters of TE buffer.
Prepare, more sample for accurate pipetting and loading (may be 30 microliters instead of 20 microliters. Dilute accordingly.
2. The GC basepairs are more stronger than AT basepairs due to presence of three hydrogen bonds than two hydrogen bonds. It is difficult to break a GC basepair bonds than AT base pair. Addition of 3 GC base pairs at 3’ end will ensure increase specificity of primers due to stronger bonding. However, more than 3 G and C will make it difficult to denature the primers during PCR.