Pre lab for lab 7
what can affect the Ct value?
1) Biological differences in your sample 2) difference in various preparation step the Ct value might be affected because of the florescent emission which is affected by PH and salt concentration -reaction values is the ratio of reporter dye to your passive reference dye. reaction efficiency;- this depends on the master key performance , the assay and the sample quality. usually the 90-110 efficiency is okay. in order to determine efficiency run serial 5 log dilutions and conduct R^2 tests. the R^2 value should be above 0.99
Chloroform extractions?What is the purpose of a chloroform extraction? Which phase of the extraction should you keep and which one should you discard?
Among the different way of cleaning RNA/DNA chloroform extraction is the best way. the phenol is buffer saturated phenol which around 72% phenol and 28% water. Phenol is a weak acid but when working with RNA or DNA, it brings it PH to the target either acidic to the RNA and basic to DNA. Phenol/choroform is buffer saturated phenol and chloroform and it is usually 1:1 for DNA extraction. the density of the buffer saturated phenol is lower than water. the chloroform is usually used after the phenol/chloroform cleaning is done. while following the procedure - it is better when a fresh sample is used. phenol solubilizes the molecule. proteins because of a lot of hydrophobic side chains they are flipped on the hydrophobic side. phenol by itself retains 10-15 % of water.
what are the house keeping genes (GAPDH)
It is an internal control gene. the house keeping gene amplification might be used as a way to estimate the quantity of the target gene.
Metaphor agarose: how is this agarose different from the agarose we have been using in our other electrophoresis protocols? Why do we need to use it for this experiment?
Metaphor agarose provides twice the resolution capability than a normal agarose. it has an intermediate melting tempreture..this gel can be used to resolve DNA fragments for PCR and RT-PCRs, if their size difference is 2%.if the fragmanets difference is less than 1% then they can be resolved in a 1.5 hr. it has a high resolution seperate 20-800 bp -recovery of fragments under 800bp -very good analysis of PCR products -
qRT-PCR: Describe the purpose and mechanism of qPCR. What are the different ways qPCR results can be measured and analysed? What is Ct (Cq)?
PCR is a very powerful technique that allows us to analyze and edit DNA. there are more than 50 ways to carry out PCR. qPCR as the name incdicated is a PCR technique that allows you to qunatify DNA. it has several applications like cancer diagnostics, food safety and public health, clinical quantification and genomic and genotyping. when performing qPCR amplification of the target PCR is done by attaching florescent probes or DNA dyes inorder to measure the quantitative information of the DNA which makes it better than a normal DNA. it measures the amount of nucleic acid present during each amplification process. the higher the florescent signal the more PCR product there is. however, even though it has a lot of advantages. it also has several drawbacks.for example if there is inconsistent amplification efficiencies of primers then it reduces the quantification efficiencies. it requires an inclusion of an external calibrator. qRT-PCR (quantitative reverse transcription-polymerase chain reaction)- it is a very good technique for detection the messengeral RNA. this is because it is sensitive enough to quantify RNA even from a single cell. this whole process depends on reverse transcription. therefore, the process needs to be optimal. the reason for that is RNA template might contain inhibitors like fatty acid, alcohol and phenol left after the process of extraction. this results in a very unreliable result of quantification. therefore, it is very improntant to choose the right DNA clean up kit. the better way to avoid this problem is to follow a two step. the two step is very advantageous because 1) reduced primer-dimer formation the qPCR includes continuous incubation. there is a technique of detecting primer dimer in qrt-PCR using a system called SYBRgreen(Which is a double stranded binding dye) . it is very bad if there is a production of primer dimer because it reduces the yield and inaccurate estimation. even a small production of primer dimer is very bad because it will be amplified by the DNA polymerase and even with the reverse transcriptase. therefore, instead of using a one step reaction, using a two step reaction is better in minimizing the qrt-PCR.this process helps in drastically reducing the formation of primer dimer. the common procedure of this technique includes initial denaturation-->cycling (Denaturation, annealing , extension) ---> repeat one application of the qPCR is it can be used to analyze the genomic DNA . one of the most common application of this process is gene expression analysis. as mentioned above the mRNA can be quantified using the one step or two step technique.
Relative quantification and comparative Ct method
endogenous control- GAPDH are evaluated
qPCR standard curve, Provide an example and understand how to make one.
even thought the qPCR technique seems like a simple, straight forward technique. it needs to have a good control inorder to have an accurate result. one of the control that is used is the qPCR standard curve. the researcher needs to make sure that the Ct value reflects the reality. the primers need to be tested first, if that step is skipped the result might not be accurate. even though the melt curve of the primer is nice and it has only a single peak it does not mean that the DNA is usable. therefore, it is very important to first test it with qPCR standard curve. how is it done? you first make the qPCR quantify different amount of the same DNA. for example test five concentrations in 1;5 dilution factor. to be more accurate do sequential dilution and pipet the same amount of DNA. use water as a negative control and that is to detect if there is any contamination in the reaction. it is important to nano drop your DNA and make sure that it has a good quality A260/A280 ratio. there are qPCR softwares that are used to calculate the efficiency of the reaction. the most acceptable is between 90-110 with a slope of 3.3 for the 100%. iyou first make the qPCR quantify different amount of the same DNA. for example test five concentrations in 1;5 dilution factor. to be more accurate do sequential dilution and pipet the same amount of DNA. use water as a negative control and that is to detect if there is any contamination in the reaction. it is important to nano drop your DNA and make sure that it has a good quality A260/A280 ratio. there are qPCR softwares that are used to calculate the efficiency of the reaction. the most acceptable is between 90-110 with a slope of 3.3 for the cause of unproprtional standard curve is not only inefficient primer. if the target DNA is expressed poorly it might help to increase the amount of DNA that is used.. your sample can be done on crude lysated instead of purifyed RNA sample. the standard curve is also important in telling you the appropriate amount of DNA to use in the next experiment.
Cq
for every individual cycle they are characterized by the cycles at which the flourecent rises from the threshold. if the amplification process is observed in the early cycles that means the starting material is abundant and the Cq (quantification cycle) is higher.
Expected result
https://www.researchgate.net/figure/Typical-Standard-curve-of-GAPDH-and-Tg-BULLET-mRNA-established-with_fig1_8219053 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3595253/ http://mcb.asm.org/content/29/8/2308.full https://www.thermofisher.com/order/catalog/product/4309155
what are the qPCR requirements
inorder to initiate the target qPCR we need a very few copies of target nucleic acid. it is advised to start with a minimum required amount of template inorder to achieve accurate quantification. when starting with RNA ,primer design and DNAse 1 treatment is important in reducing signals that are caused by contamination it is very important to be very careful when designing high quality primers. the probe is used as mentioned earlier in the amplification detection method. the appropriate type should be selected to be very efficient in this technique. the master mix of a PCR/qPCR contains dATP, dCTP, dGTP, dTTP. some mixes replae the dTTP with dUTP incubation of these mixes with UNG prevents contamination. to be effective PCR lab uses dUTP. Mgcl2 is very important in the reverse transcription, taq DNA polymerase and other activity. majority of the master mixes contain this compound. for the success of this technique a reverse transcriptase that has a high yield of cDNA is very important. when performing single step qRT-PCR it is better to use a high tempreture increases the specificity of this reaction. when doing two step it is better to make sure that the cDNA produced is linear and the selection of the best polymerase activity is very important in this whole technique. Buffers- are reaction mixtures and they contain dNTP, taq DNA polymerase, MgCl2 and stabilizers. there are different types of buffers and they are dependent on manufacturers. it is advantageous to buy these reaction mixtures because it increases consistency and convenience because it reduces the amount of pipe ting we have to do., decreases the chance of error and contamination.
Phusion polymerase: how is this polymerase different from the Taq we have been using and why do we need to use this particular polymerase for this experiment?
it follows the exonuclease activity and over take it . these enzyme have been said to be really high performance from multiple experiments and they enable high fidelity result. this DNA polymerase is made from a DNA binding domain attached to proof reading polymerase. they are tolerent of different inhibitors. it allows high amplification with minimal optimization. some of the characteristics of this polymerase includes -high fidelity -fewer reaction failure -improved yields -high speed , therefore, shorter reaction time when amplifying a DNA sequence it is important to keep the sequence especially when cloning. even if one nucleotide is wrong then that might result in the formation of a wrong protein at the end. this affects the way the proteins fold and perform their functions. the reason why it is chosen over a taq polymerase is because the error rate is 50 fold lower than a taq polymerase and 6 fold lower than the pfu DNA polymerase
PAM sequence is the protospacer adjacent motif
it is a 2-6 base pairs that immediately follows after the DNA sequence which is targeted by the Cas9 sequence in CRISPR. it is a component of the virus or anything that is trying to invade the bacteria not the component of the bacteria itself. in order for the Cas9 to succesfully cleave the DNA sequence it needs to be followed by the PAM. it is used to identify the bacteria's DNA from other DNAs. therefore, it prevents the CRISPR from being damaged. the Cas9 nuclease attaches itself to the guide RNA which guides it to the invading sequence. the invading sequence will contain a PAM and therefore it will be cut.
What is cDNA
it is a complementary DNA which is manufactured from mRNA. it is synthesized in a reaction and it is catalyzed by reverse transcriptase and DNA polymerase enzyme. this DNA is used to clone eukaryotic genes in prokaryots.
Cas9 enzyme:
it is a genomic editing meaning it is a technology that gives scientists the ability to change a DNA. the CRISPR-Cas9 is a faster and most effective than other genomic editing methods.is an RNA guided DNA endonuclease enzyme associated with the CRISPR. it unwinds foreign DNA and it interrogates. if the DNA substrate is complementary to guide DNA then the Cas9 cleaves the invading DNA. in the lab this technique works by the guide RNA attaching itself to a specific target sequence of a DNA in a genome and also to the Cas9 enzyme. the modified RNA detects the DNA sequence and the Cas9 enzyme is used to cut the DNA at a target position. then once it is cut the researchers use the cells ability to repair itself to add or delete a piece of DNA.this editing technology is very important in treating different human diseases.
normal pCR, standard PCR
it is semi quantitative, because we have to look at the end product. etbr florescent estimation, there is lack of specificity. it binds to any double bonded DNA. you get wrong start and dimer primer , a lot of things bind with the etbr florescent. qpcr is better because it is more specific. it has more problems. DNA synthesis is measured at every specific cycle by fourecent . you can give a number to it. qPCR- different types of methods used. two types of method -using general probe using syber green, a probe that florecents at a specific wavelength. the advantage is it works for everything and it is not expensive -not specific and it binds with any double stranded DNA. becuase of the negative side people develop double dye probe, they work in TaqMAN Mol beacon satellite -double dye probe are more specific. you make the oligo and you couple probes on both side. flourophores , one is called reporter- releases an emission quancher-absorbs the emission the 5' exonuclease cleaves the reporter side of the probe. the release of the reporter at a specific wavelength and that is what your measuring.
HiScribe T7 RNA synthesis kit
it is used for in vitro transcription of RNA using t7 RNA, This kit is used to produce many kinds of RNA. RNA that is produced using this technique is very important for RNA structural and functional studies, ribozyme chemistry, micro array analysis and others. the kit is enough for 50 reaction if around 20 ul is used. each standard yields around 180 ug of RNA from 1ug control template. each kit can yield around 9mg of RNA. for 32p labeling , the kit contains enough reagents for 100 reactions of 20ul each. linearized plasmid and highest purity is very important for successful use of this kit. this is because the purity of the plasmid is important for the transcription yield. the higher the purity the higher the yield of the DNA. inorder to produce an RNA of a certain length it is important to use a plasmid that is completly linearized. even though there are several choices of enzymes to use, it is recommended to use restriction enzymes that form a blunt end. therefore, after linerization it is recommended to purify a DNA using a phenol or chloroform extraction.
Components of CRISPR/Cas9
it uses non specific endonuclease to cut the genomic site and a small RNA to guide the process. it is guided to the target DNAs by two RNAs crRNA and tracr RNA. the guide RNA contains 20 nucleotides target sequence that directs the cas9 to a specific genomic sequence. the crispr protein has the advantage to bind to a guide RNA, bind to a target DNA with the help of the guide DNA and cleave a target DNA resulting in a double stranded break (DBS) The Cas9 through a knockdown and other experiments was shown to be a key player in CRISPR mechanisms. this mechanism is very important to target genes in many cell lines and organisms in human, zebra and other animals.
what is a Cq , Ct value-->threshold cycle
qRT-PCR is very good technique of detecting the amount of mRNA, it is sensitive enough to detect an RNA just from one cell. As the name indicates this technique depends on reverse transcription. This reaction needs to be done carefully because the RNA template might contain fatty acid, alcohol and phenol left after the process of extraction, which results in a very unreliable result of quantification. Therefore. it is very important to use the right DNA clean up kit. if the Ct value is below 29 cycles that means the sample has abundant nucleic acid if it is above 38 cycles indicate minimal account. reading the florescent amount at the beginning is better and accurate than reading at the end. the reason for that is because when reaching the end point accumulated end point, inactivated polymerase, and limiting reagents create a lot of variations. After about 15 cycles the amount of background florescent can be detected. it will be almost a straight line from the zero cycle point then when it reaches the threshold level it will be a little above this line. the exact point where this happens can be calculated by using a computer software. the computer will also calculate the sample runs. the Ct value will be at the threshold interaction.
B6/SCID mice
severe combined immune deficiency -it is the inability of the immune system to sustain an appropriate immune system. it is characterized by the absence of T cells and B cells. this is caused by mutation on chromosome 16 which has enzyme that is used for DNA repair. for that reason they are an ideal model for cell transfer/tissue transplant and they can be used to study the immune system in the lab. some of them might develop partial immune reactivity. these mice are used in studying the normal and abnormal lymphocytes development and function. therefore, they are a used to study the normal and abnormal lymphocyte development and function.
Bioneer Accuprep PCR Kit
this is another DNA purification kit that purrifies upto 10ul of DNA in 5 minutes. DNA from 10-100bp can be purified effectively using this technique. the recovery yield for this kit exceeds 80%. in this technique removal of oil is not that important. this kit works by adsorption of DNA in a glass filter. chaopatric salts are using to make the attachment of the DNA better. the adsorption mechanism is very selective because other compounds like protein is not adsorbed and they pass through the glass. therefore, when the whole solution is washed it gets rid of the protein and everything else. once the DNA is purified it can be cloned and other biological application.
O'Gene Ruler 1 kbp ladder
this ladder is used in quantification of wide variety of double stranded DNA fragment in agarose gel. it is made from different mixtures that are purified through chromatography. it is already premixed with an orange loading gel. this ladder is usually used when analyzing small DNA fragments. it is used for DNA sizing and approximate quantification when sharp bands are needed it is a bright reference band it can directly be loaded and it is stable for a very long time (around 6months) -it is supplied with a loading dye
the two step or one step quantifying technique of the qRT-PCR
two step-in this technique the q-PCR ad the reverse transcription technique is done in two separate tubes. usually the starting material for this process is RNA. one step- the qPCR and the reverse transcription technique is done in one tube. this simplifies the experimental technique. this technique also has a lower chance of contamination because there is no following step to handle it.
what are we gonna do in lab (cumsky)
we do a standard curve GAPDH- it is an important step 6- glycolysis- it is abundent in cells that make energy. because they metabolize glucose. standard curve on mouth GAPDH-plasid on the experiment you are going to measure GPDH RNA for mouse. we will be provided with mouse RNA in lab and we are going to use GAPDH and sybr green to measure the amount of GAPDH present. when you see the graph on the slide showing the graph of the cycle number and Rn, it is just a normal DNA of interest. around the certain cycle you are going to take the RNA and make CDNA form it and then you do qPCR on it. the lower the amount you start with you see a cycle at the higher number. after doing the standard curve. melting curve is done, the sample is melted. this is to make sure sometimes when you see the melting curve ??? it might show you that there are other compounds other than the GAPDH
