12.8. Using the same PCR conditions and primers re-amplify fragment for ca. 25 cycles - more cycles may be necessary for weak or small fragments.
12.9. The amplified fragment may be purified after separation on a 1.2% agarose gel or cloned directly.
LSB Buffer 10.0 mM TRIS pH 7.5
1.0 mM EDTA
100.0 mM LiCl
HSB Buffer 10.0 mM TRIS pH 7.5
1.0 mM EDTA
1.0 M LiCl
20.0 % Ethanol
13. AseI/TaqI PCR primers used for cDNA-AFLP
§ POLY D[T]25V, BIOTINYLATION AT 5' END
5’- TTTTTTTTTTTTTTTTTTTTTTTTV
Total number of bases is: 26.
DNA sequence composition: 0 A; 0 C; 0 G; 25 T; 1 OTHER;
Sequence name: OLIGO00
-----------------------------------------
§ TOP STRAND ADAPTOR - ASEI
5’- CTCGTAGACTGCGTACC
Total number of bases is: 17.
DNA sequence composition: 3 A; 6 C; 4 G; 4 T; 0 OTHER;
Sequence name: OLIGO41/ A-Top
-----------------------------------------
§ ADAPTOR BOTTOM STRAND - ASEI
5’- TAGGTACGCAGTC
Total number of bases is: 13.
DNA sequence composition: 3 A; 3 C; 4 G; 3 T; 0 OTHER;
Sequence name: OLIGO42 / A-bottom
-----------------------------------------
§ ASEI - + 0
5’- CTCGTAGACTGCGTACCTAAT
Total number of bases is: 21.
DNA sequence composition: 5 A; 6 C; 4 G; 6 T; 0 OTHER;
Sequence name: OLIGO43 / A00
-----------------------------------------
§ ASEI, + 2 – AA
5’- GACTGCGTACCTAATAA
Total number of bases is: 17.
DNA sequence composition: 6 A; 4 C; 3 G; 4 T; 0 OTHER;
Sequence name: OLIGO44 / A11
-----------------------------------------
§ ASEI, + 2, - AC
5’- GACTGCGTACCTAATAC
Total number of bases is: 17.
DNA sequence composition: 5 A; 5 C; 3 G; 4 T; 0 OTHER;
Sequence name: OLIGO45 / A12
-----------------------------------------
§ ASEI, + 2 - AG
5’- GACTGCGTACCTAATAG
Total number of bases is: 17.
DNA sequence composition: 5 A; 4 C; 4 G; 4 T; 0 OTHER;
Sequence name: OLIGO46 / A13
-----------------------------------------
§ ASEI, + 2 - AT
5’- GACTGCGTACCTAATAT
Total number of bases is: 17.
DNA sequence composition: 5 A; 4 C; 3 G; 5 T; 0 OTHER;
Sequence name: OLIGO47 / A14
-----------------------------------------
§ ASEI, + 2 - CA
5’- GACTGCGTACCTAATCA
Total number of bases is: 17.
DNA sequence composition: 5 A; 5 C; 3 G; 4 T; 0 OTHER;
Sequence name: OLIGO48 / A15
-----------------------------------------
§ ASEI, + 2 - CC
5’- GACTGCGTACCTAATCC
Total number of bases is: 17.
DNA sequence composition: 4 A; 6 C; 3 G; 4 T; 0 OTHER;
Sequence name: OLIGO49 /A16
-----------------------------------------
§ ASEI, + 2 - CG
5’- GACTGCGTACCTAATCG
Total number of bases is: 17.
DNA sequence composition: 4 A; 5 C; 4 G; 4 T; 0 OTHER;
Sequence name: OLIGO50 / A17
-----------------------------------------
§ ASEI, + 2 - CT
5’- GACTGCGTACCTAATCT
Total number of bases is: 17.
DNA sequence composition: 4 A; 5 C; 3 G; 5 T; 0 OTHER;
Sequence name: OLIGO51 / A18
-----------------------------------------
§ ASEI, + 2 - GA
5’- GACTGCGTACCTAATGA
Total number of bases is: 17.
DNA sequence composition: 5 A; 4 C; 4 G; 4 T; 0 OTHER;
Sequence name: OLIGO52 / A19
-----------------------------------------
§ ASEI, + 2 - GC
5’- GACTGCGTACCTAATGC
Total number of bases is: 17.
DNA sequence composition: 4 A; 5 C; 4 G; 4 T; 0 OTHER;
Sequence name: OLIGO53 / A20
-----------------------------------------
§ ASEI, + 2 – GG
5’- GACTGCGTACCTAATGG
Total number of bases is: 17.
DNA sequence composition: 4 A; 4 C; 5 G; 4 T; 0 OTHER;
Sequence name: OLIGO54 / A21
-----------------------------------------
§ ASEI, +2 - GT
5’- GACTGCGTACCTAATGT
Total number of bases is: 17.
DNA sequence composition: 4 A; 4 C; 4 G; 5 T; 0 OTHER;
Sequence name: OLIGO55 / A22
-----------------------------------------
§ ASEI, +2 - TA
5’- GACTGCGTACCTAATTA
Total number of bases is: 17.
DNA sequence composition: 5 A; 4 C; 3 G; 5 T; 0 OTHER;
Sequence name: OLIGO56 / A23
-----------------------------------------
§ ASEI, +2 - TC
5’- GACTGCGTACCTAATTC
Total number of bases is: 17.
DNA sequence composition: 4 A; 5 C; 3 G; 5 T; 0 OTHER;
Sequence name: OLIGO57 / A24
-----------------------------------------
§ ASEI, +2 - TG
5’- GACTGCGTACCTAATTG
Total number of bases is: 17.
DNA sequence composition: 4 A; 4 C; 4 G; 5 T; 0 OTHER;
Sequence name: OLIGO58 / A25
-----------------------------------------
§ ASEI, + 2 - TT
5’- GACTGCGTACCTAATTT
Total number of bases is: 17.
DNA sequence composition: 4 A; 4 C; 3 G; 6 T; 0 OTHER;
Sequence name: OLIGO59 / A26
-----------------------------------------
§ ADAPTOR, TOP STRAND-TAQI
5’- GACGATGAGTCCTGAC
Total number of bases is: 16.
DNA sequence composition: 4 A; 4 C; 5 G; 3 T; 0 OTHER;
Sequence name: OLIGO61 / T-Top
-----------------------------------------
§ ADAPTOR, BOTTOM STRANS - TAQI
5’- CGGTCAGGACTCAT
Total number of bases is: 14.
DNA sequence composition: 3 A; 4 C; 4 G; 3 T; 0 OTHER;
Sequence name: OLIGO62 / T-bottom
-----------------------------------------
§ TAQI, + 0
5’- GACGATGAGTCCTGACCGA
Total number of bases is: 19.
DNA sequence composition: 5 A; 5 C; 6 G; 3 T; 0 OTHER;
Sequence name: OLIGO63 / T00
-----------------------------------------
§ TAQI, + 2 - AA
5’- GATGAGTCCTGACCGAAA
Total number of bases is: 18.
DNA sequence composition: 6 A; 4 C; 5 G; 3 T; 0 OTHER;
Sequence name: OLIGO64 / T11
-----------------------------------------
§ TAQI, + 2 - AC
5’- GATGAGTCCTGACCGAAC
Total number of bases is: 18.
DNA sequence composition: 5 A; 5 C; 5 G; 3 T; 0 OTHER;
Sequence name: OLIGO65 / T12
-----------------------------------------
§ TAQI, + 2 - AG
5’- GATGAGTCCTGACCGAAG
Total number of bases is: 18.
DNA sequence composition: 5 A; 4 C; 6 G; 3 T; 0 OTHER;
Sequence name: OLIGO66 / T13
-----------------------------------------
§ TAQI, +2 - AT
5’- GATGAGTCCTGACCGAAT
Total number of bases is: 18.
DNA sequence composition: 5 A; 4 C; 5 G; 4 T; 0 OTHER;
Sequence name: OLIGO67 / T14
-----------------------------------------
§ TAQI, +2 - CA
5’- GATGAGTCCTGACCGACA
Total number of bases is: 18.
DNA sequence composition: 5 A; 5 C; 5 G; 3 T; 0 OTHER;
Sequence name: OLIGO68 / T15
-----------------------------------------
§ TAQI, +2 - CC
5’- GATGAGTCCTGACCGACC
Total number of bases is: 18.
DNA sequence composition: 4 A; 6 C; 5 G; 3 T; 0 OTHER;
Sequence name: OLIGO69 / T16
-----------------------------------------
§ TAQI, +2 - CG
5’- GATGAGTCCTGACCGACG
Total number of bases is: 18.
DNA sequence composition: 4 A; 5 C; 6 G; 3 T; 0 OTHER;
Sequence name: OLIGO70 / T17
-----------------------------------------
§ TAQI, +2 - CT
5’- GATGAGTCCTGACCGACT
Total number of bases is: 18.
DNA sequence composition: 4 A; 5 C; 5 G; 4 T; 0 OTHER;
Sequence name: OLIGO71 / T18
-----------------------------------------
§ TAQI, +2 - GA
5’- GATGAGTCCTGACCGAGA
Total number of bases is: 18.
DNA sequence composition: 5 A; 4 C; 6 G; 3 T; 0 OTHER;
Sequence name: OLIGO72 / T19
-----------------------------------------
§ TAQI, +2 - GC
5’- GATGAGTCCTGACCGAGC
Total number of bases is: 18.
DNA sequence composition: 4 A; 5 C; 6 G; 3 T; 0 OTHER;
Sequence name: OLIGO73 / T20
-----------------------------------------
§ TAQI, +2 - GG
5’- GATGAGTCCTGACCGAGG
Total number of bases is: 18.
DNA sequence composition: 4 A; 4 C; 7 G; 3 T; 0 OTHER;
Sequence name: OLIGO74 / T21
-----------------------------------------
§ TAQI, +2 - GT
5’- GATGAGTCCTGACCGAGT
Total number of bases is: 18.
DNA sequence composition: 4 A; 4 C; 6 G; 4 T; 0 OTHER;
Sequence name: OLIGO75/ T22
-----------------------------------------
§ TAQI, +2 - TA
5’- GATGAGTCCTGACCGATA
Total number of bases is: 18.
DNA sequence composition: 5 A; 4 C; 5 G; 4 T; 0 OTHER;
Sequence name: OLIGO76 / T23
-----------------------------------------
§ TAQI, +2 - TC
5’- GATGAGTCCTGACCGATC
Total number of bases is: 18.
DNA sequence composition: 4 A; 5 C; 5 G; 4 T; 0 OTHER;
Sequence name: OLIGO77 / T24
-----------------------------------------
§ TAQI, +2 - TG
5’- GATGAGTCCTGACCGATG
Total number of bases is: 18.
DNA sequence composition: 4 A; 4 C; 6 G; 4 T; 0 OTHER;
Sequence name: OLIGO78 / T25
-----------------------------------------
§ TAQI, +2 - TT
5’- GATGAGTCCTGACCGATT
Total number of bases is: 18.
DNA sequence composition: 4 A; 4 C; 5 G; 5 T; 0 OTHER;
Sequence name: OLIGO79 / T26
