Please help, will give a thumbs up Lab 4 Protocol #2: Polymerase Chain Reaction
ID: 262924 • Letter: P
Question
Please help, will give a thumbs up
Lab 4 Protocol #2: Polymerase Chain Reaction (PCR) 1. In your PCRs, you will be using two primers specific for the pET41-EGFP reco nt plasmids. One of the primers, Primer 1 is specific for the EGFP insert; the other primer, Primer 2, is specific for a region of the pET-41 vector that flanks the EGFP insert. 2. Your DNA templates" in PCR will be the three plasmids you isolated last week (ie. your minipreps), plus a "positive control" pET41/EGFP recombinant plasmid that we have isolated, giving you a total of four templates. You will also run a "negative control reaction, where no template DNA is included. Think about what this negative control tells you. 3. Here are the "stock" reagents used for your reactions 10X PCR Buffer (alrcady contains Mg) 10 mM dNTPs 10 uM of Primer 1 primer 10 uM of Primer 2 primer 5 Units/ ul Taq polymerase Plasmid DNA preps (you know their concentrations from last week) Nuclease-free H20 should have a total volume of 20 ul. Below, on the left are the final concentrations, or where appropriate, the final amounts, of each reagent you'l need to have in each reaction Before you come to class, calculate the appropriate volumes of each stock reagent and water you'll add together for each reaction. CALCULATIONS lur S millimi Reagent: Final C Volume of Stock Reagent to Add ul of 10 X PCR Buffer ul of 10 mM dNTPs ul of 10 uM Primerl ul of 10 uM of Primer 2 ul of "0.5 ng/ul" plasmid DNA* ul of 5U/ul Taq polymerase ul U. X PCR Buffer 2. 800 uM dNTPs 2 #x 20 (3). 0.5 uM Primer 1 ML C0.5 uM Primer 2 . 20 E. 2 ng plasmid DNA* ° 2.5 Units Taq polymerase Nuclease-free H20 (to 20 ul final) * Based on the individual concentrations of each of your miniprepes that you quantitated last week, you need to calculate a) How many ul of each miniprepep wi ill you need to make a 500uL dilution to 0.5ng/ul? This calculations (you are creating dilutions of your template) b) Then you can calculate how many uL of your 0.5ng/ul working dilutions you need to add to each PCR reaction as template, and fill in the above table accordinglyExplanation / Answer
Answer:
1. Point just gives the information of your primers which going to used in PCR. Here plasmid is pET41. eGFP was cloned in this plasmid. The clone was represented as pET41+eGFP= pET41-EGFP (Recombinant vector).
2. In general, PCR used to know the presence of desired gene/ fragment in given sample (DNA/ plasmid). Here you have three plasmids, we don't have information about plasmids which contained EGFP gene with pET41 background. Means we have to identify the pET41-eGFP among three isolate plasmids. These results have to confirmed by comparing with known results. So known pET41-EGFP used as a positive control. pET41 vector alone (without eGFP) used as template in the negative control. In case of nontemplate control, nuclease-free water used instead of DNA/Plasmid in PCR reaction.
3. Here they mentioned stocks contraction of components which used in PCR. These stocks have to dilute to working concentrations, according to our reaction set up. Here N1V1=N2V2 Formula.
4. 1.) stock of PCR buffer is 10X (N1), working concentration is 1X (N2) in 20ul reaction volume (V2), substitute the values above formula.
10X V1= 1X20
V2= 2ul
2.) 1.6ul 3.) 1ul 4.) 1ul 5.) 4ul 6.) 0.5ul Taq polymerase 7.) 9.9 ul nuclease-free water
* You need to quantify the isolated plasmid samples to know the concentration. Here we used two methods to quantify. 1) Nano drop (Based on absorbance): Here the instrument will give direct concentration values in ng/ul. Eg: 500ng/ul or 800 ng/ul. 2) Through run in agarose gel, comparing the band intensity with band intensity of known sample concentration.
* Based on the original concentration we have to dilute to 0.5ng/ul in 500ul.
eg: N1= 500ng/ul; V1 =X ; N2= 0.5ng/ul; V2= 500ul By substituting in N1V1=N2V2 formula.
V1= 0.5 ul. Take 0.5ul from original stock and add 499.5ul of nuclease-free water to make up to 500ul. Then the concentration is 0.5ng/ul (working stock).
You have working stock (0.5ng/ul), from this have to add into PCR reaction, it should be 2ng.
In working stock each ul having 0.5ng plasmid. PCR required 2ng, So have to add 4 ul from working stock (0.5ng/ul).