Documents about Pcr Products
418_assign_5
Cornell, BIO 4180
Excerpt: ... Name _ Exercise 5 (30 points) due Friday April18, 2008 BioMI 418 Microbial Ecology Questions for lecture notes. These questions focus on some things you should consider about community fingerprinting methods. Use your class notes to answer the following: 1) There are many methods used by microbial ecologists to generate a community profile or fingerprint, using PCR products generated from amplification of genes from DNA extracted from an environmental sample. a) Name one fingerprinting method that allows you to recover a band (PCR product), PCR amplify it and determine its sequence. b) Name one community fingerprinting method that does not allow you to recover DNA bands for further analysis. 2) Review your notes on carbon isotopes. a) 14 C What does the number "14" indicate about this isotope (compared to 12 C)? b) What is meant by the term "stable" isotope? Were going to focus on Marine Microbiology over the next few lectures but since we can't ...
|
|
MBIO 3410 lecture Nov 6 2007
Manitoba, MBIO 3410
Excerpt: ... of a heterozygote will yield two different sized DNA fragments (156 and 138 bp) 9 10 Cloning PCR Products It is possible to clone PCR products directly The terminal transferase activity of Taq DNA polymerase adds a template-independent A residue to the 3'-end of PCR products forming a single 3'-A overhang called TA cloning These can be ligated to a linearized `T-vector', which has single 3'-T overhangs on both ends The complementarily between the PCR product 3'-A overhangs and vector 3'-T overhangs aids efficient ligation 11 RTPCR Reverse transcription polymerase chain reaction (RTPCR) has been devised as a method of RNA amplification and quantification after its conversion to DNA The technique consists of two parts: the synthesis of DNA from RNA by reverse transcription (RT) subsequent amplification of a specific DNA molecule by PCR The RT reaction uses: an RNA template a primer dNTPs buffer Reverse transcriptase enzyme Generation of a single-stra ...
|
|
Expt7protocol
UCLA, HC 70
Excerpt: ... L - L 50 L - L - L 50 L - L - L 50 L - L - L 50 L 2 L - L 50 L - L 2 L 50 L 48 L 2 L - L - L - L 48 L - L 2 L - L - L 48 L - L - L 2 L - L 48 L - L - L - L 2 L 48 L - L - L - L - L 48 L - L - L - L - L 17. Carry out PCR reactions with the RT-PCR program containing the following profile: 1 cycle of 96 oC, 3 min. " 40 cycles of 94 oC, 10 sec./60 oC, 30 sec./72 oC, 45 sec. " 1 cycle of 72 oC, 4 min. " 4 oC, !. 18. Prepare 100 mL of 1.5% agarose gel in 1X TAE buffer as usual (Use a 20-tooth comb). Note: The percentage of agarose gels depends on the difference in size of two PCR products . If there is at least 100-bp difference between two PCR products , then use a 1% agarose gel. However, if there is 50-100 bp difference between two PCR products , then use 1.5-2% agarose gel. For example, the size of PCR products is 0.6 kb and 0.55 kb for the control and gene A, respectively. The ...
|
|
AppNote119-optimase
BYU, WAVEASSAY 2
Excerpt: ... Figure 1 trace A shows the results of analysis of PCR product produced using the Optimase Polymerase for which the average percentage of heteroduplex DNA was found to be 7.9 1.02 %. Traces obtained for polymerases that induced the incorporation of high numbers of errors during amplification showed a detrimental effect on data analysis. Figure 1 shows data traces obtained for Optimase Polymerase (trace A) and an alternative polymerase (trace B) showing heteroduplex formation in 7.3% and 22.1% of PCR products respectively. The effect of these mis-incorporations is clearly visible and at high levels the quality of data acquired using the WAVE System may be impaired. Results from the analysis of all polymerases tested are presented in Figure 2. The data determined in this study correlate well with the relative error incorporation rates that have been shown for these polymerases in other studies (26), confirming that analysis of the percentage of heteroduplex fragments gives an equivalent measure of replicatio ...
|
|
FS2006-Review_exam4-Lec38-40
Michigan State University, MMG 301
Excerpt: ... ISA assays used for how is PCR used in diagnostics; what two methods are used to monitor the production of PCR products ...
|
|
Expt7protocol
UCLA, HC 70
Excerpt: ... mL - mL 2 mL - mL 4 48 mL - mL - mL - mL 2 mL 5 48 mL - mL - mL - mL - mL 6 48 mL - mL - mL - mL - mL (Positive) (Negative) Mmix Leaf +RT Leaf -RT Silique +RT Silique -RT 0.2 ng/mL Genomic DNA Water Total Volume - mL - mL 50 mL - mL - mL 50 mL - mL - mL 50 mL - mL - mL 50 mL 2 mL - mL 50 mL - mL 2 mL 50 mL 17. Carry out PCR reactions with the RT-PCR program containing the following profile: 1 cycle of 96 oC, 3 min. 40 cycles of 94 oC, 10 sec./60 oC, 30 sec./72 oC, 45 sec. 1 cycle of 72 oC, 4 min. 4 oC, . 18. Prepare 100 mL of 1.5% agarose gel in 1X TAE buffer as usual (Use a 20-tooth comb). Note: The percentage of agarose gels depends on the difference in size of two PCR products . If there is at least 100-bp difference between two PCR products , then use a 1% agarose gel. However, if there is 50-100 bp difference between two PCR products , then use 1.5-2% agarose gel. For example, the size of PCR products is 0.6 kb and 0.55 ...
|
|
Protocol 4.2
Maryland, BSCI 412
Excerpt: ... the red band has migrated at least 60% of the available length, turn off the power supply and disconnect the leads. 4. Photographing DNA in agarose WEARING GLOVES, remove gel tray from box YOUR TA WILL: The TA will then place the gel in the photographic chamber. Close the door. Using the white light source position the gel in the image zone on the computer screen with the wells visible. Turn on the UV light source. While looking at the image on the computer screen, adjust light exposure and zoom (should be already focused). The bands in the 1 kb ladder should be clearly visible. Print out photograph and check image again the printout. If not satisfactory then adjust and print out again. Dispose of gel in the indicated ethidium bromide waste container. Check if you have an 1kb band for each of the PCR product. If so, please continue with the next step. If you have zero or one out of the two PCR, let your TA know. Exo-AP treatment Similar to Project 2.6, you must treat PCR products with Exo-AP to remove pr ...
|
|
lec6
Sveriges lantbruksuniversitet, MBB 308
Excerpt: ... You will get a product with Us in it. If you pretreat future PCR reactions with Uracil N-glycosylase (the repair enzyme which removes Us after deamination of DNA), this chops up the PCR product, so it wont be amplified in the next reaction (handout). 1. 2. Errors from Taq polymerase: No proof-reading, so amplified products may not represent the true sequence of the template. So either direct DNA sequencing of a mixed population of PCR products (the average sequence should be OK), or clone several different representative products and sequence, to confirm. Or, choose another thermostable enzyme which edits and corrects. 5 3. Taq pol also leaves a 3 A overhang, so that the PCR product is more difficult to clone. Either: (i) polish the ends with T4 DNA polymerase or (ii) choose a special vector with matching T tail, OR (iii) choose another thermostable polymerase which doesnt leave a 3 A overhang, or (iv) design special PCR primers which contain extra sequences and restric ...
|
|
class7-ppt
University of Illinois, Urbana Champaign, CPSC 265
Excerpt: ... Bacterial cloning Especially of PCR product DNA PCR recap PCR gel product 1 Cloning Cloning is the way in which we can take a single molecule, and make lots of bacterial cells that contain an identical molecule. These cells are clones, hence the name This used to be the only way to amplify DNA. It is still by far the most accurate. Plasmid vectors circular, autonomous bacterial DNA 2 Cloning PCR products When we amplify DNA using PCR, it is often necessary to clone this DNA We do this in order to replicate it without errors Also, by cloning a protein coding sequence into E. coli, we can then produce the protein in the bacterium. The vector is made with a T overhang Taq polymerase leaves an A overhang Taq is the thermostable DNA polymerase from Thermus aquaticus we used for PCR. When Taq synthesizes a new strand, it always puts an extra A at the end This can be useful, but note: other polymerases do not do this, only Taq polymerase 3 ...
|
|
lambdaINTpcr
Ole Miss, CHEM 472
Excerpt: ... Separate Phage and Bacterial DNA genomes before integration AttP Lysogen structure after recombination AttB AttL AttR d1 The goals of the web exercise are to use 4 primers and various templates to determine unique PCR product sizes for each templ ...
|
|
class7
University of Illinois, Urbana Champaign, CPSC 265
Excerpt: ... Bacterial cloning Especially of PCR product DNA PCR recap PCR gel product Cloning Cloning is the way in which we can take a single molecule, and make lots of bacterial cells that contain an identical molecule. These cells are clones, hence the name This used to be the only way to amplify DNA. It is still by far the most accurate. Plasmid vectors circular, autonomous bacterial DNA Cloning PCR products When we amplify DNA using PCR, it is often necessary to "clone" this DNA We do this in order to replicate it without errors Also, by cloning a protein coding sequence into E. coli, we can then produce the protein in the bacterium. The vector is made with a "T" overhang Taq polymerase leaves an "A" overhang Taq is the thermostable DNA polymerase from Thermus aquaticus we used for PCR. When Taq synthesizes a new strand, it always puts an extra "A" at the end This can be useful, but note: other polymerases do not do this, only Taq polymerase Electroporation i) When the elec ...
|
|
BIOS816-lecture5
UNL, BIOS 816
Excerpt: ... n OK again In Results tab, check Save PCR Product(s) as DNA Molecule(s) to DB and Save Primer(s) as Oligo(s) to DB Click Run Click Save Report and save the report to your desktop as `Multiplex 1' Click Close Report When prompted to save both PCR products and Oligos to the Vector NTI database leave both options click OK Close the Lists dialog box Return to the Vector NTI Explorer and examine the sequences in the Cluster 1 subbase in the DNA/RNA molecules database and the PCR primers in the Cluster 1 subbase in the Oligo database 21 04/14/09 Multiplex PCR Multiple Templates Open the DNA molecule YPL262W Select the FUM 1 CDS in the graphics pane Choose List | Clear Multiplex PCR List Choose List | Multiplex PCR List Click Add, check Add Whole Sequence then click OK Repeat this process for the following regions from 3 other DNA molecules: i) PYK2 CDS from YOR347C; ii) CIT1 CDS from YNR001C; iii) MLS1 CDS from YNL117W Click Run Click the Results tab and change the prefixes of both subbas ...
|
|
primers
UVA, DTA 4
Excerpt: ... PCR primers for KO, tagging, detection * amplification of pRS400a (KanMX) or pRS403 (HIS3) for KO: 5'- X40 5' end YFG- GAT TGT ACT GAG AGT GCA CC -3' 5'-X40 3' end YFG- CTG TGC GGT ATT TCA CAC CG -3' -> ~1.6 kb KanMX PCR product, ~2.1 kb HIS3 PCR pro ...
|
|
CYP1Aflowchart
Kenyon, BIOL 64
Excerpt: ... intact RNA. Step 3: Total cDNA synthesis. The total RNA will be used as a template for reverse transcriptase (RT), an enzyme that catalyzes the synthesis of complementary DNA (cDNA) from RNA templates. The RT reaction will be primed using random hexamers. Step 4: PCR with degenerate primers. cDNA synthesized in the RT reaction will be used as a template for the polymerase chain reaction (PCR). You will use PCR primers designed based on regions of conserved amino acids identified in the alignment of CYP1A protein sequences from a number of species. These primers must be degenerate, i.e. contain multiple nucleotide possibilities at positions where an amino acid was encoded by more than one codon. At positions where 3 or 4 nucleotides are possible, Inosine can be used, since it can form base pairs with A, C, G, or T. Step 5: Analysis and Gel purification of the PCR products . We will identify PCR products suitable for further study based on their size. Proper size is predicted based on the positions of primer ...
|
|
PCRGel
UCSD, BICD 123
Excerpt: ... bp Lane 1 2 3 4 5 6 7 12000 2000 1650 1000 850 650 500 400 300 200 100 AP3 PCR gel. Lane 1 is the 1 kb plus ladder, lanes 2 4 are the PCR product from wildtype DNA extracts, lanes 5 7 are the PCR products from DNA extracts from the ap3 mutant. Lane 8 is the PCR product using water as a negative control. H2O (negative control) 8 Ladder (1 kb plus) wildtype ap3 ...
|
|
SubCloning
UMass (Amherst), TWIKI 4
Excerpt: ... %META:TOPICINFO{author="ktheis" date="1181075697" format="1.1" reprev="1.2" version="1.2"}% %META:TOPICPARENT{name="KarstenPrimerDesign"}% -+ Subcloning exercise -+ Your task The _E.coli_ genomic DNA contains the gene for a DNA repair protein, [http ...
|
|
Figure6 Legend
University of Texas, JLV 1212
Excerpt: ... 97 Figure 6. Alignment of the 82-bp fragments from RT- PCR products , A2-1 and A2-4, corresponding to sequences used by Misof and Wagner to identify allelic variants. ...
|
|
MAss22007AS
Laurentian, BIOL 3200
Excerpt: ... Biology 3200 Minor Assignment 2 This assignment is due in class on April 10. It is worth 4% of your final mark for this course. Your assignment must be typed double-spaced in a 12-point font on white paper. It must not exceed 5 pages in length includ ...
|
|
primer_key
UCLA, CHEM 154
Excerpt: ... You are studying a novel protein. The gene encoding this protein of interest is written in Figure 1 of your supplementary material. Your goal is to perform site-directed mutagenesis on this gene of interest, clone the mutated version of the gene into ...
|
|
Garen
UCLA, HC 70
Excerpt: ... Arabidopsis Thaliana A Study of Genes and Embryo Development By Garen Polatoglu A T 1 G 2 2 1 9 0 Gene Gene Length: 1511 bp Translated Region: 786 bp PCR Product: 1004 bp Encoded Protein Size: 261 amino acids Putative AP2 domain contain ...
|
|
FactorIX_Wet_Lab
Lycoming, SRV 2
Excerpt: ... ion Primer annealing Primer extension2 Stage 3 - 1 cycle standard denaturation Primer annealing Primer extension 1 min. @ 94oC 1 min. @ 55oC 1.5 min. @ 72oC 1 min. @ 94oC 1 min. @ 55oC 10 min. @ 72oC Notes: 1 To improve specificity, template DNA concentration, annealing temperature and Mg2+ concentration may be varied. 2 1 minute extension time should be used for each kbp of product expected. C. Prepare 1% agarose gel To save time during next week's lab, today we will prepare the agarose gel to analyze the PCR products , wrap it in plastic and store it until next week. 1. Securely tape the ends of a gel tray such that a small amount of tape is on the underside of tray. Place tray on a sheet of plastic wrap in case of spillage. Align comb in tray parallel with and 1-2 cm from the end of the tray. 2. Add an appropriate amount of agarose (0.4g) to 40 mL water in a 125 mL erlenmeyer flask, heat mixture in microwave on high setting for 45 sec - 1.5 min, or until mixture begins to boil. 3. Using a folded paper ...
|
|
FragAnalysis
Washington, FISH 543
Excerpt: ... amount of ladder. The injection parameters of the instrument are standardized and will not be altered to accommodate specific samples. PCR Protocol The PCR protocol used to generate the labeled fragments is at the discretion of the client. We suggest designing a thermocycler protocol for 25-35 cycles that gives a large yield of PCR product when run on a slab gel. Primers Only one of a pair of primers for a single locus should be labeled The internal size standard is labeled with ET-ROX so the dyes available for your samples are FAM, HEX, and NED (or TAMRA). NED is sold only by Perkin-Elmer but the others are available from Operon. If you plan on multiplexing, it is helpful to design the primers to have approximately the same annealing temperature. Sample Preparation Most of our in-lab genotypers use an Ethanol Precipitation (protocol available upon request) to clean up their PCR products . If you plan to multiplex, it is easiest to first combine your PCR products and then clean them up together. After clean-up ...
|