What is Splinkerette?
This can be used to map the insertion sites of transposable elements (and often works when other methods fail, such as inverse PCR).
What is ligation mediated PCR?
Ligation-mediated polymerase chain reaction (LM-PCR) is a genomic analysis technique for determination of (1) primary DNA nucleotide sequences (2) cytosine methylation patterns (3) DNA lesion formation and repair, and (4) in vivo protein–DNA footprints1,2,3,4.
How does Tn5 Tagmentation work?
Illumina developed the tagmentation protocol, in which a modified Tn5 enzyme cuts double-stranded DNA and concurrently ligates the linker sequences that are required for Illumina sequencing to both ends.
What is AFLP marker?
Summary. Amplified fragment length polymorphism (AFLP) is a PCR-based technique that uses selective amplification of a subset of digested DNA fragments to generate and compare unique fingerprints for genomes of interest.
What is mini primer PCR?
Here we show that a new PCR method using an engineered polymerase and 10-nucleotide “miniprimers” expands the scope of detectable sequences beyond those detected by standard methods using longer primers and Taq polymerase.
What is the purpose of Tagmentation?
Tagmentation is the initial step in library prep where unfragmented DNA is cleaved and tagged for analysis. On-bead tagmentation library prep uses bead-linked transposomes for a more uniform tagmentation reaction compared to in-solution tagmentation reactions.
What does Tagmentation mean?
tagmentation (uncountable) (genetics) A process, in the analysis of DNA, in which double-stranded DNA is cleaved and tagged.
What is the difference between RFLP and AFLP?
Both RFLPs and AFLPs involve differences in the lengths of DNA fragments. The two techniques differ because RFLPs are revealed by digestion with restriction enzymes, whereas AFLPs involve both digestion with restriction enzymes and a selective amplification step.
Why adapters are used in AFLP?
(A) Adapters are designed to be complementary to each other and to allow sticky-end ligation with PstI restriction fragments. (B) Tagged fragments represent the DNA template in the following PCR reaction. (C) Selective primers used in the final amplification step.
What are adapters in Illumina sequencing?
The adapters contain the sequencing primer binding sites, the index sequences, and the sites that allow library fragments to attach to the flow cell lawn. Libraries prepared with Illumina library prep kits require adapter trimming only on the 3′ ends of reads, because adapter sequences are not found on the 5′ ends.
What is Nextera XT Tagmentation?
The Nextera XT DNA Sample Preparation Kit uses an engineered transposome to simultaneously fragment and tag (“tagment”) input DNA, adding unique adapter sequences in the process. A limited-cycle PCR reaction uses these adapter sequences to amplify the insert DNA.
What is Tagmentation with Tn5?
Tagmentation-based library construction, using the Tn5 transposase, is efficient for generating sequencing libraries but currently relies on undisclosed reagents, which severely limits development of novel applications and the execution of large-scale projects.
What is Adaptor ligation in NGS?
What is Adapter Ligation Technology? Ligation technology is used to construct NGS libraries for sequencing. The process uses an enzyme to connect specialized adapters to both ends of DNA fragments. An ‘A’- base is added to the blunt ends of each strand, preparing them for ligation to the sequencing adapters.
What is universal primer?
Universal primers are complementary to nucleotide sequences that are very common in a particular set of DNA molecules and cloning vectors. Thus, they are able to bind to a wide variety of DNA templates.
What does AFLP stand for?
Amplified fragment length polymorphism (AFLP) is a PCR-based technique that uses selective amplification of a subset of digested DNA fragments to generate and compare unique fingerprints for genomes of interest.
Does splinkerette-PCR favour shorter products?
Since splinkerette-PCR is a multiplex-PCR reaction it will generally favour the amplification of shorter PCR products from within the pool of insertion sites found within a sample.
What is splinkerette PCR used for in Drosophila?
This can be used to map the insertion sites of transposable elements (and often works when other methods fail, such as inverse PCR). * Potter CJ, Luo L. Splinkerette PCR for Mapping Transposable Elements in Drosophila. PloS ONE. 2010;5 (4):e10168; *corresponding author.
How can I make a splinkerette-adaptor for PCR reactions using SOC media?
This adapter mix can be stored indefinitely at −20°C and will provide enough splinkerette-adaptor for 100 splinkerette-PCR reactions. SOC medium To 950 mL double-distilled water, add 20 g Bacto-tryptone, 5 g Bacto-yeast extract, 0.5 g NaCl and 2.5 mL of 1 M KCl.
Is there a supplementary table for splinkerette-PCR products for 454 sequencing?
Supplementary Table 1:10 bp sequences suitable for ‘barcoding’ splinkerette products for 454 sequencing. These sequences facilitate the computational ‘demultiplexing’ of pooled splinkerette-PCR products following 454 sequencing. Click here to view.
https://www.youtube.com/watch?v=h7_me6Tq3c4