2. AGAAGATAAGTAGATGGCAGTCCGTACGGTCAT
TCTTCTATTCATCTACCGTCAGGCATGCCAGTA
AGAAGATAAGTAGATGGCAGTCCGTACGGTCATTGACTGACTTGACTGACGTCGGTACTTGACT
Partial cleavage at A Partial cleavage at T Partial cleavage at G Partial cleavage at C
AGAAGATAAGTAGATGGCA
AGAAGATAAGTAGA
AGAAGATAAGTA
AGAAGATAA
AGAAGATA
AGAAGA
AGAAGATAAGTAGAT
AGAAGATAAGT
AGAAGAT
AGAAGATAAGTAGATGG
AGAAGATAAGTAGATG
AGAAGATAAGTAG
AGAAGATAAG
AGAAG
AGAAGATAAGTAGATGGCAGT AGAAGATAAGTAGATGGCAG AGAAGATAAGTAGATGGCAGTCC
AGAAGATAAGTAGATGGCAGTC
AGAAGATAAGTAGATGGC

3. AGAAGATAAGTAGATGGCA
AGAAGATAAGTAGA
AGAAGATAAGTA
AGAAGATAA
AGAAGATA
AGAAGA
AGAAGATAAGTAGAT
AGAAGATAAGT
AGAAGAT
AGAAGATAAGTAGATGG
AGAAGATAAGTAGATG
AGAAGATAAGTAG
AGAAGATAAG
AGAAG
AGAAGATAAGTAGATGGCAGT
AGAAGATAAGTAGATGGCAG
AGAAGATAAGTAGATGGCAGTCC
AGAAGATAAGTAGATGGCAGTC
AGAAGATAAGTAGATGGC
A G C T
A
A
A
A
A
A
T
T
T
G
G
G
G
G
T
G
C
C
C

4. OH
AGAAGATAAGTAGATGGCAGTCCGTACGGTCATTGACTGACTTGACTGACGTCGGTACTTGACT
CCATGAACTGA
AGAAGATAAGTAGATGGCAGTCCGTACGGTCATTGACTGACTTGACTGACGTCGGTACTTGACT
AGCCATGAACTGA
AGCT
A
DNA polymerase
AGAAGATAAGTAGATGGCAGTCCGTACGGTCATTGACTGACTTGACTGACGTCGGTACTTGACT
ACTGACTGCAGCCATGAACTGA
AGAAGATAAGTAGATGGCAGTCCGTACGGTCATTGACTGACTTGACTGACGTCGGTACTTGACT
AACTGACTGCAGCCATGAACTGA
AGAAGATAAGTAGATGGCAGTCCGTACGGTCATTGACTGACTTGACTGACGTCGGTACTTGACT
ACTGAACTGACTGCAGCCATGAACTGA
AGAAGATAAGTAGATGGCAGTCCGTACGGTCATTGACTGACTTGACTGACGTCGGTACTTGACT
ACTGACTGAACTGACTGCAGCCATGAACTGA
AGAAGATAAGTAGATGGCAGTCCGTACGGTCATTGACTGACTTGACTGACGTCGGTACTTGACT
ACTGCAGCCATGAACTGA

5. Manual DNA sequencing
1- DNA preparation:
- High quality
- At least two microgram DNA
- The more the merrier
2- Single strand DNA is ready to go, double strand DNA needs denaturation:
- Heat and annealing with the primer (on ice-water)
- Using one micro of 1N NaOH and neutralize with HCl
Plasmid Phagemid
M13
3- Labeling, DNA polymerase, little dNTPs, Radioactive dATP (S35, P33, P32)
4- Prepare 4 tubes, each contain dNTP plus one of the ddNTP, label the tubes
AGC or T. Transfer one fourth of the mixture from (3) to the A tube, one
fourth to the G tube, one fourth to the C tube and one fourth to the T tube

6. A tube
AGCT
A
DNA polymerase
5- Stop the reaction in the four tubes using loading buffer containing
formamide, heat up and load the four reaction in adjacent four lanes on
polyacrylamide gel that contain 50% urea
6- Run the gel at high voltage, dry, expose to X-ray film and develop the film
Have a nice reading

7. 1-DNA amount and quality. Columns, impurities
2-Double Strand DNA, full denaturation, excess primer
3-Single stand DNA, too many steps in preparation
4-PCR products, fast re-annealing of the fragment, excess primer, fast
annealing
A G C T
5-GC compression: Secondary structure in the
newly synthesized strand
Gel temperature
Nucleotides analogues, 7-diaza

8. A G C T
6-Secondary structure in the template
Extension temperature (40 T7 polymerase)
Chasing with dNTP and terminal transferase
7-gel problems:
Smiling
Gel compression at the top
A G C T
Aluminum plates
Gradient gel
Gradient buffer

9. - Four reaction in four separate tubes
- Very low amount of DNA (0.1 picomole)
1picomole of double strand in ng = size of DNA x 0.66
- Linear increase of the newly synthesized DNA
-Less sensitivity to impurities
-Full denaturation of DNA
-Less secondary structure in the template DNA