Sequencing Lab:  Notes on protocol

 

The sequencing lab will be performed using the Thermo Sequenase II dye terminator cycle sequencing premix kit.  The instructions for use of this kit have been placed on reserve in the library under the name “Sequencing lab protocol”.  This kit uses “Thermo Sequenase II”.  This is a modified Taq polymerase which has the 5'-3' exonuclease activity deleted, as well as point mutations that allow it to add dideoxy nucleotides and dITP to a sequence.  (Wild type Taq polymerase does not add modified nucleotides very efficiently.)  Termination of primer extension is produced by 4 different dideoxy nucleotides, each attached to a different fluorescent dye.  This means that all 4 nucleotides can be identified in the same lane of a gel by differences in fluorescence. 

 

Each group will prepare 1 sequencing reaction (see page 7 of “Sequencing lab protocol”).  Note that each reaction costs $10, and that there is no extra reaction mix – be very careful to pipette the correct amount.  The template DNA being sequenced will be pPBH plasmid (pBluescript containing a cloned histone gene insert).  The primers used will be derived from the regions of the T3 and T7 promoters flanking the pBluescript multiple cloning site.  Each pair of groups on one side of a bench should decide which will use T3 or T7 – the results for both primers can then be shared between groups, and analysis of sequence from both ends of the cloned insert will be performed.

 

T3 primer (5'-3')

ATTAACCCTCACTAAAG

 

T7 primer (5'-3')

AATACGACTCACTATAG

 

After all 3 groups have prepared their reaction mixes, the samples will be run on the automated sequencing equipment in the Davidson Lab (SSB 6150).  If you wish to see the equipment in action, you may visit SSB 6150 after 14:00 on Friday afternoon.

 

Modifications to Protocol

 

Materials (per group)

 

Thermo Sequenase II reagent mix A+B (premixed)        4 µl

5 µM primer (T3 or T7) in ddH2O                                   1 µl

0.067 µg/µl DNA template (pPBH) in ddH2O               15 µl

                                                                                        20 µl

2 µl sodium acetate/EDTA buffer

95 % ethanol

70 % ethanol

4 µl Formamide loading dye (see handout for MSDS information)

 

1 0.5 ml Eppendorf tube

 

Methods

 

Follow steps 2-5 of the “Preparation of sequencing reaction” protocol.  Note that reagents A and B have already been mixed.  Follow steps 1-6 in the “preparation for gel loading” protocol, and hand in your sample, which will be frozen until Friday.  Note that you will have to calculate the amount of 95 % ethanol to add to produce a final concentration of 75 % in your sample.

 

Study questions (due next week)

 

1.         Calculate the amount (in µg) of DNA template and the amount of primer (in nmol) present in the reaction mix.  Show sample calculations.

 

2.         Why are T7 Sequenase and wild type Taq polymerases not suitable for cycle sequencing?

 

3.         Why were the template and primers prepared in ddH2O?

 

4.         What are the advantages of using dye terminator and cycle sequencing?

 

Lab report (due in final week of labs)

 

The long format lab report will be based on the 3 sequencing labs in this week and the following 2 weeks.  A template for the lab report will be posted on the website.