Your Federal Quarterly Tax Payments are due April 15th Get Help Now >>

AFLP Data Sheets and Other Documents by qdj95169

VIEWS: 77 PAGES: 5

									                           AFLP Data Sheets and Other Documents

1.   Restriction Digestion/Restriction-Ligation Reactions
2.   Pre-selective/Selective Amplifications
3.   Codes for Primer Combinations
4.   Restriction-Ligation Reaction Constituents Table
     AFLP RESTRICTION DIGESTION AND RESTRICTION-LIGATION REACTIONS

    Project(s):_________________________________________________       Date:____/____/______

RESTRICTION ENZYME DIGESTIONS
Samples:
              1      2     3                  4        5         6         7       8
Row 1
Row 2
Row 3

Total # Samples:______________         Reaction volume per sample: 10 l plus DNA

Restriction Enzyme Master Mix Constituents:          Affix gel image below:
Constituents:      Per Sample Total in Mix
MseI enzyme        0.01 l     __________
EcoRI enzyme       0.05 l     __________
MseI buffer        1 l        __________
EcoRI buffer       1 l        __________
Water              8 l        __________

Thermal cycler program: “aflp-digestion”

Comments on digestions:


RESTRICTION-LIGATION REACTIONS
Samples:
               1     2     3                  4        5         6         7       8
Row 1
Row 2
Row 3

Total # Samples:______________         Reaction volume per sample: 11 l

Enzyme Master Mix (1 l/sample):              Restriction-Ligation Master Mix (4.5 l/sample):
Constituents:     Per Sample Total in Mix     Constituents:     Per Sample     Total in Mix
T4 ligase buffer  0.1 l       __________     T4 ligase buffer 1 l            __________
NaCl (0.5M)       0.1 l       __________     NaCl (0.5M)       1 l           __________
BSA (1mg/ml)      0.05 l      __________     BSA (1mg/ml)      0.5 l         __________
MseI enzyme       0.05 l      __________     MseI adaptors     1 l           __________
EcoRI enzyme      0.25 l      __________     EcoRI adaptors 1 l              __________
T4 DNA ligase     0.33 l      __________
Water             0.22 l      __________
Thermal cycler program: “aflp-digestion”
                                                                                                 3


        AFLP PRE-SELECTIVE AND SELECTIVE AMPLIFICATION REACTIONS

    Project(s):_________________________________________________       Date:____/____/______

PRE-SELECTIVE AMPLIFICATIONS
Samples:
               1     2      3                  4        5        6           7        8
Primer Pair
Samples
Primer Pair
Samples
Primer Pair
Samples

Total # Samples:______________           Reaction volume per sample: 10 l
Pre-Selective Amp Master Mix Constituents:
                                   Row A #:_____       Row B #:_____   Row C #:_____
Constituents:         Per Sample Total in Mix          Total in Mix    Total in Mix
AFLP core mix         7.5 l       __________          __________      __________
Pre-sel primer pairs 0.5 l        __________          __________      __________
(Diluted res-lig rxn) (2 l)
Thermal cycler program: “aflp-preselective”
Comments on pre-selective amplifications (gel image taped on back):


SELECTIVE AMPLIFICATIONS
Samples:
              1      2                3        4        5        6           7        8
Primer Pair
Samples
Primer Pair
Samples
Primer Pair
Samples

Total # Samples:______________           Reaction volume per sample: 10 l
Selective Amp Master Mix Constituents:
                                          Row A #:_____ Row B #:_____            Row C #:_____
Constituents:              Per Sample Total in Mix          Total in Mix         Total in Mix
AFLP core mix              7.5 l         __________        __________           __________
MseI (Primer-CXXX)         0.5 l         __________        __________           __________
EcoRI (Dye-primer-AXX) 0.5 l             __________        __________           __________
(Diluted pre-sel amp)      (1.5 l)
Thermal cycler program: “aflp-selective”
                                                                     4


                    CODES FOR AFLP PRIMER COMBINATIONS

EcoRI\MseI:   CAA    CAC    CAG    CAT    CTA    CTC     CTG   CTT
Fam-AAC        B1     B2     B3     B4     B5     B6      B7    B8
Fam-ACA        B9    B10    B11    B12    B13    B14     B15   B16
Ned-AAC        Y1     Y2     Y3     Y4     Y5     Y6      Y7    Y8
Ned-ACC        Y9    Y10    Y11    Y12    Y13    Y14     Y15   Y16
Ned-AGC       Y17    Y18    Y19    Y20    Y21    Y22     Y23   Y24
Joe-AAG        G1     G2     G3     G4     G5     G6      G7    G8
Joe-AGG        G9    G10    G11    G12    G13    G14     G15   G16
Joe-ACG       G17    G18    G19    G20    G21    G22     G23   G24
                                                                                                                       5


                                       AFLP RESTRICTION-LIGATION REACTION CONSTITUENTS
                             ENZYME MASTER MIX                                   RESTRICTION-LIGATION MASTER MIX
#       T4 buffer    NaCl    BSA   MseI   EcoRI    T4 ligase    water  T4 buffer   NaCl   BSA     MseI adpt    EcoRI adpt
    1            0.1   0.1    0.05  0.05     0.25          0.33   0.22           1      1   0.5             1                1
    2            0.2   0.2     0.1   0.1      0.5          0.66   0.44           2      2     1             2                2
    3            0.3   0.3    0.15  0.15     0.75          0.99   0.66           3      3   1.5             3                3
    4            0.4   0.4     0.2   0.2        1          1.32   0.88           4      4     2             4                4

    5          0.5     0.5    0.25   0.25    1.25         1.65    1.1           5      5    2.5            5                 5
    6          0.6     0.6     0.3    0.3     1.5         1.98   1.32           6      6      3            6                 6
    7          0.7     0.7    0.35   0.35    1.75         2.31   1.54           7      7    3.5            7                 7
    8          0.8     0.8     0.4    0.4       2         2.64   1.76           8      8      4            8                 8

 9             0.9     0.9    0.45   0.45    2.25         2.97   1.98           9      9    4.5            9                 9
10               1       1     0.5    0.5     2.5          3.3    2.2          10     10      5           10                10
11             1.1     1.1    0.55   0.55    2.75         3.63   2.42          11     11    5.5           11                11
12             1.2     1.2     0.6    0.6       3         3.96   2.64          12     12      6           12                12

13             1.3     1.3    0.65   0.65    3.25         4.29   2.86          13     13    6.5           13                13
14             1.4     1.4     0.7    0.7     3.5         4.62   3.08          14     14      7           14                14
15             1.5     1.5    0.75   0.75    3.75         4.95    3.3          15     15    7.5           15                15
16             1.6     1.6     0.8    0.8       4         5.28   3.52          16     16      8           16                16

17             1.7     1.7    0.85   0.85    4.25         5.61   3.74          17     17    8.5           17                17
18             1.8     1.8     0.9    0.9     4.5         5.94   3.96          18     18      9           18                18
19             1.9     1.9    0.95   0.95    4.75         6.27   4.18          19     19    9.5           19                19
20               2       2       1      1       5          6.6    4.4          20     20     10           20                20

21             2.1     2.1    1.05   1.05    5.25         6.93   4.62          21     21   10.5           21                21
22             2.2     2.2     1.1    1.1     5.5         7.26   4.84          22     22     11           22                22
23             2.3     2.3    1.15   1.15    5.75         7.59   5.06          23     23   11.5           23                23
24             2.4     2.4     1.2    1.2       6         7.92   5.28          24     24     12           24                24

								
To top