By Juris Steinbergs, Aleksander Tsimanis (auth.), Professor Dr. Valdis Berzins (eds.)
This handbook introduces the reader to bacic equipment utilized in the isolation, cloning and research of genetic fabric. The protocols comprise RT-PCR amplification, gene cloning, hybridization research and sequencing of nucleic acids, PCR-based site-specific mutagenesis, research of protein DNA-specific interplay, cell-free protein synthesis and product electrophoretic and immunological research. each one protocol comprises brief heritage info, a close description of the mandatory fabrics, step by step tactics, a troubleshooting advisor and beneficial sensible hints.
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Extra info for Basic Cloning Procedures
The insertion of mutations at other sites inside of the amplified gene sequence is therefore not possible. Using mutagenesis by overlap extension allows the introduction of sequence alterations at any position within the DNA region, not just at the ends of the sequence. This method makes possible the insertion of specific mutations into the nucleotide sequence directly from the cloned gene into its original vector with approx. 100 % efficiency using a few simple steps (Ho et al. 1989). The principle of the method is the generation of two peR fragments with overlapping ends that can be effectively fused by recombining them in a subsequent peR reaction.
Primer to template mole ratio should be 1:1 to 1:5. 38 E. JANKEVICS GATCGATC Fig. 2. Parallel bands are present in all four lanes close to primer and they are shifted by 'half-step'. Labeling reaction artifacts. Arrows indicate nonspecific bands • A background of even but high intensity is obtained, such that specific bands become hard to distinguish - Contaminated DNA preparation, try control DNA. • One or more of the tracks is too faint or too short - This is due to incorrect dNTP/ddNTP ratios; tracks that are too faint are usually caused by not having enough dideoxynucleoside triphosphate present, while short tracks are caused by having too much present.
Centrifuge for 5 min in a micro centrifuge to remove the remaining cells. Transfer supernatant to a fresh microcentrifuge tube. 9. l of PEG/NaCl to the micro centrifuge tube. Mix well, then let stand for 15 min at ambient temperature. Alternatively, leave overnight at 4°C. 10. Centrifuge for 5 min, discard supernatant. 11. Centrifuge for 2 min. Carefully remove all remaining traces of PEG with a glass capillary. Wipe off with a tissue any traces of PEG on the mouth of the tube. 12. l of TE buffer.