Navigation Links
Optimizing the Direct Amplification of Missing cDNA 5' Ends,,, Using the Eppendorf Mastercycler gradient

Optimizing the Direct Amplification of Missing cDNA 5' Ends
Using the Eppendorf Mastercycler gradient

Ulrich Genschel
Institute of General Botany, University of Hamburg Christian Rohrer, Eppendorf AG, Hamburg

Introduction

In many research projects, cDNA isolation forms the basis for detailed analyses at the molecular level. However, when cDNA libraries are screened using conventional plaque hybridization techniques, cDNA clones with an incomplete 5' end are frequently obtained. Since a complete cDNA, or at least a complete open reading frame, is required for many applications (such as expression of functional proteins or sequence-based prediction of sub-cellular location), the missing 5' sequence has to be obtained at a later point in time. However, isolation of full-length cDNAs by plaque screening is extremely labor-intensive when the sought-after cDNA is present at low abundance in a given library.

Direct amplification

One alternative is direct PCR amplification of the missing sequence using the cDNA library as template [1,2]. In this case, a vector primer (VP) and a gene-specific primer (GP) are created as shown in the scheme in Fig. 1. As all cDNA clones in the library act as a template for the vector primer, nonspecific products are formed very easily in this PCR reaction. It is therefore important to use highly-specific primers and to ensure that reaction conditions are as stringent as possible, with one crucial factor being the setting of the annealing temperature. An example of this strategy is given below.


'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Optimizing Transfection Conditions for Studying Signal Transduction Pathways
2. Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer
3. Optimizing electroporation parameters
4. Optimizing Büchi® Rotary Evaporator Applications
5. Optimizing electroporation parameters
6. Optimizing Büchi® Rotary Evaporator Applications
7. Optimizing Bchi Rotary Evaporator Applications
8. Optimizing electroporation parameters
9. Optimizing electroporation parameters
10. Optimizing electroporation parameters
11. Optimizing electroporation parameters
Post Your Comments:
(Date:7/10/2014)... QC (PRWEB) July 10, 2014 EvoDerma ... and convenience. This rejuvenating device now comes with a second ... develops over time. , The new cup is thinner on ... design works to target rough, thin and uneven surfaces on ... firm cup for a stimulating treatment on areas such as ...
(Date:7/10/2014)... July 10, 2014  Franciscan St. Anthony ... capnography for respiratory monitoring outside the operating ... leaders in embracing state-of-the-art patient safety technology. ... are breathing and can alert medical caregivers ... the amount of carbon dioxide the patient ...
(Date:7/10/2014)... 2014  Kainos Capital, a firm specializing in acquiring ... it has acquired the Slim-Fast brand from Unilever. Unilever ... of the transaction were not disclosed. Slim-Fast ... that markets ready-to-drink shakes, powders, bars and snacks to ... and in the United Kingdom ...
(Date:7/10/2014)... July 10, 2014 Research and Markets ... the "International Photonic Integrated Circuit (Monolithic Integration, ... 2019" report to their offering. ... concept of photonic integration traces its roots in ... promise of photonic integration went unexplored and unfulfilled ...
Breaking Biology Technology:Introducing a New Bonus Treatment Cup for EvoDerma’s NOOME Anti-Aging Motion 2Franciscan St. Anthony Health-Crown Point Underscores Patient Safety Commitment Through Expanded Use of Capnography 2Kainos Capital Acquires Slim-Fast From Unilever 2International Photonic Integrated Circuit (Monolithic Integration, Hybrid Integration, Module Integration) Market - Forecasts to 2019 2
... Annualized Relapse Rate and was Well Tolerated in MS Patients -- PARIS, August 30, 2010 ... ... ... ,; var first_result; // Results are keyed by longUrl, so we need to grab the first one. ...
... Inc. (OTC Bulletin Board: GOVX ), a biotechnology ... announced the results of its Annual Shareholders Meeting, held on ... seven nominees for directors of the company to serve until ... are duly elected or appointed and qualified. They are: ...
... Bioheart, Inc., (OTC Bulletin Board: BHRT ) ... with stem cell therapy on the first two congestive ... Angeles Tijuana through its Center of Excellence, Regenerative Medicine ... an agreement to establish a Center of Excellence for ...
Cached Biology Technology:Teriflunomide Significantly Reduced Annualized Relapse Rate and was Well Tolerated in MS Patients 2Teriflunomide Significantly Reduced Annualized Relapse Rate and was Well Tolerated in MS Patients 3Teriflunomide Significantly Reduced Annualized Relapse Rate and was Well Tolerated in MS Patients 4Teriflunomide Significantly Reduced Annualized Relapse Rate and was Well Tolerated in MS Patients 5Teriflunomide Significantly Reduced Annualized Relapse Rate and was Well Tolerated in MS Patients 6Teriflunomide Significantly Reduced Annualized Relapse Rate and was Well Tolerated in MS Patients 7Teriflunomide Significantly Reduced Annualized Relapse Rate and was Well Tolerated in MS Patients 8Teriflunomide Significantly Reduced Annualized Relapse Rate and was Well Tolerated in MS Patients 9GeoVax Labs Inc. Announces Results of 2010 Annual Stockholders Meeting 2GeoVax Labs Inc. Announces Results of 2010 Annual Stockholders Meeting 3GeoVax Labs Inc. Announces Results of 2010 Annual Stockholders Meeting 4Bioheart Reports Significant Improvements in Heart Failure Patients from Center of Excellence Program 2Bioheart Reports Significant Improvements in Heart Failure Patients from Center of Excellence Program 3Bioheart Reports Significant Improvements in Heart Failure Patients from Center of Excellence Program 4
(Date:7/11/2014)... team of researchers, including scientists from the Max Planck ... reported a major step in understanding photosynthesis, the process ... the oxygen in its atmosphere and which is therefore ... , The researchers report the first direct visualization ... the step in which a specific protein complex, photosystem ...
(Date:7/11/2014)... stem cells can stimulate neurogenesis in the brain of ... and improve tissue and function injury under the condition ... therapeutic effect of adipose-derived stem cells (ADSCs) transplantation in ... injury and neurogenesis in the brain of AD mice. ... Sciences, Tsinghua University, China transplanted ADSCs into the hippocampus ...
(Date:7/11/2014)... DUBLIN , July 11, 2014 ... the "International Biometrics Technology Market - Industry Analysis ... report to their offering. ... attacks and crimes has created a need for high ... methods used for personnel authentication such as username and ...
Breaking Biology News(10 mins):A first direct glimpse of photosynthesis in action 2International Biometrics Technology Market - Industry Analysis Size Share Growth Trends and Forecast to 2019 2
... ARBORA common test used to determine mercury exposure from ... the toxic metal released from fillings, according to University ... fillings slowly release mercury vapor into the mouth. But ... of whether this exposure presents a significant health risk ...
... process that releases the energy in coal without burning ... passed a milestone on the route to possible commercial use, ... Energy & Fuels describes results of a successful 200-hour ... two inexpensive but highly polluting forms of coal. ...
... 2013) - House paint containing dangerous,concentrations of lead is ... and the,company is refusing to remove the paint from ... regulations to restrict the lead content of paint in,Cameroon ... of,Occupational Knowledge International (OK International) and co-author of a ...
Cached Biology News:Measuring mercury: Common test may overestimate exposure from dental amalgam fillings 2Measuring mercury: Common test may overestimate exposure from dental amalgam fillings 3US company identified as manufacture of lead paint in Africa 2