• DNA测序

• 点击：    作者：   来源： 日期：2006-11-09    本站论坛

DNA测序（主要内容如下）

·         Sequencing Gel Preparation

·         Preparation of Templates 

·         DNA Sequencing by the Dideoxy Method

·         DNA Sequencing by the Chemical Method

·         Dye Terminator DNA Sequencing

·         Construction of Nested Deletions for Sequencing

·         PCR Sequencing

·         Recipes

·         Q & A Posted in Molecular Biology Method Forum

---

Sequencing Gel Preparation

·         (NWFSC)
Gel preparation, buffer, electrophoresis...

·         (NWFSC)
Wedge gel performance without pouring wedge gels

·         (Promega)

·         (Bowtell Lab)

·        

·         (PMCI Research)

Preparation of Templates 

·         (Promega)

·         (Genetics Resources CORE Facility at Johns Hopkins U)
Detailed method for preparing PCR products for direct sequencing

·         (Genetics Resources CORE Facility at Johns Hopkins U)

·           (Genetics Resources CORE Facility at Johns Hopkins U)

DNA Sequencing by the Dideoxy Method

·         Double Stranded DNA Sequencing (Dideoxy) ( (Bowtell Lab)

• (Donis Keller Lab)
  This method is used to sequence double stranded DNA, such as a plasmid insert or purified PCR product. The dideoxy chain termination (or enzymatic) method of DNA sequencing involves the in vitro synthesis of a DNA strand by a modified bacteriophage T7 DNA polymerase (SequenaseR/USB) using a single stranded DNA template.

·         (Lazo Lab)

·         (Brent Graveley)

·         (Promega)

·         (Promega)

·         (PMCI Research)

• (Roe Lab)

                            i.           

                        ii.           

                    iii.           

                        iv.           

                            v.           

                        vi.           

                    vii.           

                viii.           

DNA Sequencing by the Chemical Method

·         Preparation of G+A Marker
Simplified method for preparing G+A ladder run along with footprinting reaction. It's much simple than the original Maxima-Gilbert sequencing reaction and works fine.

Dye Terminator DNA Sequencing

• Dye Termination Sequencing of dsDNA. (Gimila Lab)
     

·         (Whitehead Institute/MIT)

·         (Whitehead Institute/MIT)

·         (Whitehead Institute/MIT)

·         (Whitehead Institute/MIT)

·         (Whitehead Institute/MIT)

·         (PMCI Research)

·         (PMCI Research)

Construction of Nested Deletions for Sequencing

·         (Bowtell Lab)
Nested Deletions using exonuclease-III and mung bean nuclease

·         (Promega)
For generation and selection of oligonucleotide-directed mutants

·         (Promega)
For construction of plasmid or M13 subclones containing progressive unidirectional deletions of  inserted DNA, Including methods for DNA preparation, vector digestion, exonuclease deletion, ligation,  transformation...

·         (Crawford Lab)

PCR Sequencing

·         (GSCWU)
This protocol can be used instead of EXO/SAP for removing excess primers and nucleotides from PCR products before cycle-sequencing

·         (Donis-Keller lab)
This method is used to sequence directly a specific PCR product after large scale amplification. The reaction should be carried out in the usual fashion, except that after optimization of annealing temperature and other conditions in the 5 µl volume, a large scale 50 µl (10X) total reaction volume should be used to generate sufficient PCR product for sequencing.

·         (Genetics Resources CORE Facility at Johns Hopkins U)
Detailed method for preparing PCR products for direct sequencing

• 96 Well Plate Purification of PCR products
  Purification with Sepahcryl S-300 or S-500 - in filter bottom plates  96-well silent screen plate, Purification with Guanidine-HCl in MES buffer - in glass filter plates
  Purification with Guanidine-HCl in KAc buffer
    
• (Robert H. Cruickshank)

 

Recipes

• (Sequencing Group, UW)
  Recipes for gel solutions, loading buffer...