TAIL 2° REACTION PROGRAM:

Control Method: CALCULATED

1=4° for 2 min.

2=94° for 10 sec.

3=64° for 1 min.

4=72° for 2 min. 30 sec.

5=94° for 10 sec.

6=64° for 1 min.

7=72° for 2 min. 30 sec.

8=94° for 10 sec.

9=44° for 1 min.

10=72° for 2 min. 30 sec.

11=Go to step 2, for 11 more cycles

12=72° for 5 min.

13=4° forever

14=END

TAIL 3° REACTION PROGRAM:

Control Method: CALCULATED

1=4° for 2 min.

2=94° for 10 sec.

3=44° for 1 min.

4=72° for 2 min. 30 sec.

5=Go to step 2, for 19 more cycles

6=72° for 5 min.

7=4° forever

8=END

AD (Arbitrary Degenerate) Primer Sequences and Concentrations:

AD1: NGTCGASWGANAWGAA AD2: TGWGNAGSANCASAGA AD3: AGWGNAGWANCAWAGG AD4: STTGNTASTNCTNTGC AD5: NTCGASTWTSGWGTT AD6: WGTGNAGWANCANAGA

128-fold degenerate 12µM 128-fold degenerate 12µM 128-fold degenerate 12µM 256-fold degenerate 16µM 64-fold degenerate 8µM 256-fold degenerate 16µM

Stock concentrations of AD primers should be 20µM. To achieve the concentrations required for TAIL reactions, dilute in a seperate tube. The final amount of 400µL is sufficient for all 3 TAIL reactions.

64-fold degenerate Add 160µL primer and 240µL ddH2O

128-fold degenerate Add 240µL primer and 160µL ddH2O

256-fold degenerate Add 320µL primer and 180µL ddH2O



Alternate Plate Setups for TAIL If certain AD primers and/or border primers are found to produce more reliable products there is no need to use the other primers. As an example, I found the LB primer to work more often than the RB primer. Similarly, I found the AD1 and AD4 primers to generate nonspecific (vector) products at a high rate. Therefore, I designed a plate using ONLY the LB primer and AD2, AD3, AD5, and AD6 primers. This increased the maximum amount of DNA samples that I could run on one plate from 8 to 24. Obviously this can save a lot of time and materials. Here is an example of the modified plate setup.

DNA1 AD2	DNA1 AD3	DNA1 AD5	DNA1 AD6	DNA 9 AD2	DNA9 AD3	DNA9 AD5	DNA9 AD6	DNA17 AD2	DNA17 AD3	DNA17 AD5	DNA17 AD6
DNA2 AD2	DNA2 AD3	DNA2 AD5	DNA2 AD6	DNA 10 AD2	DNA10 AD3	DNA10 AD5	DNA10 AD6	DNA18 A D2	DNA18 AD3	DNA18 AD5	DNA18 AD6
DNA3 AD2	DNA3 AD3	DNA3 AD5	DNA3 AD6	DNA 11 AD2	DNA11 AD3	DNA11 AD5	DNA11 AD6	DNA19 A D2	DNA19 AD3	DNA19 AD5	DNA19 AD6
DNA4 AD2	DNA4 AD3	DNA4 AD5	DNA4 AD6	DNA 12 AD2	DNA12 AD3	DNA12 AD5	DNA12 AD6	DNA20 A D2	DNA20 AD3	DNA20 AD5	DNA20 AD6
DNA5 AD2	DNA5 AD3	DNA5 AD5	DNA5 AD6	DNA 13 AD2	DNA13 AD3	DNA13 AD5	DNA13 AD6	DNA21 A D2	DNA21 AD3	DNA21 AD5	DNA21 AD6
DNA6 AD2	DNA6 AD3	DNA6 AD5	DNA6 AD6	DNA 14 AD2	DNA14 AD3	DNA14 AD5	DNA14 AD6	DNA22 A D2	DNA22 AD3	DNA22 AD5	DNA22 AD6
DNA7 AD2	DNA7 AD3	DNA7 AD5	DNA7 AD6	DNA 15 AD2	DNA15 AD3	DNA15 AD5	DNA15 AD6	DNA23 A D2	DNA23 AD3	DNA23 AD5	DNA23 AD6
DNA8 AD2	DNA8 AD3	DNA8 AD5	DNA8 AD6	DNA 16 AD2	DNA16 AD3	DNA16 AD5	DNA16 AD6	DNA24 A D2	DNA24 AD3	DNA24 AD5	DNA24 AD6

** Note that only left border is used in the entire plate
If an alternate setup is used, remember to modify the cocktail for each reaction via the TAIL Recipe setup sheet. The recipe can be manipulated to accommaodate any number of AD primers and individual DNA samples. If the cocktail volume is greater than 1.5mL, two tubes will be needed to prepare the cocktail; divide each components' value by two and use 2 tubes.

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