Minimum GC-Rich Sequences for Overlap Extension PCR and Primer Annealing - DNA Cloning and Assembly Methods - page 173

Table 2
Nomenclature of GC-rich sequence

Name Sequence

15C 5 ′ -ccccccccccccccc-3 ′

15G 5 ′ -ggggggggggggggg-3 ′

5CGC 5 ′ -cccccgggggccccc-3 ′

5GCG 5 ′ -gggggcccccggggg-3 ′

′ ′

3CGC 5 -cccgggcccgggccc-3

3GCG 5 ′ -gggcccgggcccggg-3 ′

12C 5 ′ -cccccccccccc-3 ′

9C 5 ′ -ccccccccc-3 ′

6C 5 ′ -cccccc-3 ′

5CG12 5 ′ -cccccgggggcc-3 ′

5CG9 5 ′ -cccccgggg-3 ′

5CG6 5 ′ -cccccg-3 ′

3CG12 5 ′ -cccgggcccggg-3 ′

3CG9 5 ′ -cccgggccc-3 ′

′ ′

3CG6 5 -cccggg-3

Rule 3 . Oligonucleotides that relate to genes are also designed
to be complementary to these genes. This can be marked by
including “c” at the end of their name. For example,
pEGFP+699c indicates that the 5 end is located at position

699-bp downstream from the A of ATG of EGFP gene and that
the sequence is complementary to the EGFP coding sequence.
Rule 4 . Names can be combined according to the 5

′

′ -3 ′

sequence and joined with a hyphen. For example, 5GCG-
pEGFP+699c indicates that this oligonucleotide contains
5GCG at the 5

end, followed by the pEGFP+699c sequence.
2. The GC-rich overlap or annealing sequences are designated as
follows (Table 2 ).
3. A 0.8-2 % (w/v) agarose gel is prepared in 1× TAE, and
1-2

′

L of a DNA product was loaded.
4. Measurement of DNA concentration is important for reliable
fusion PCR. We use a fl uorometer to measure DNA concentra-
tion as it requires small amount of DNA solution and RNA
removal is not required. Usually 40-100 ng/

μ

μ L DNA is

obtained by PCR.