User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/12/11

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Cycle Sequencing Clones M13FWD

83 colonies PCR amped -> sequencing

Nanodrop Data

Sample ID Nucleic Acid Conc. Unit 260/280 260/230
clone1 20.6 ng/µl 1.59 1.1
clone2 12.8 ng/µl 1.93 1.12
clone3 8.8 ng/µl 1.46 1.21
clone4 10.2 ng/µl 1.57 1.1
clone5 21.9 ng/µl 1.49 1.14
Cycle Sequencing ' ' '
Reagents Concentration 1X 87X
BDT 0.5 43.5
M13 FWD 0.4 34.8
DMSO 0.1 8.7
5X Seq Buffer 5X 0.4 34.8
H20 3.6 313.2
PEG DNA variable 1
TOTAL 6 435
Cocktail per well = 5.0ul
PCR 96C 10 sec
50C 5 sec
60C 4 min
60 cycles

Cycle Sequencing AV10007, AV10016, AV10027, AV10153, PR10008 and 5475F + 8047R

Cycle Sequencing ' ' '
Reagents Concentration 1X 14X
BDT 0.5 7
DMSO 0.1 1.4
5X Seq Buffer 5X 0.4 5.6
H20 3.6 50.4
*Primer 15uM 0.4
*PEG DNA variable 1
TOTAL 6 64.4
Cocktail per well = 4.6ul
*add primer and PEG separately
PCR 96C 10 sec
50C 5 sec
60C 4 min
60 cycles
Primers Sequence FWD and RVS
AV10007
AV10016
AV10027
AV10153
PR10008
5475F + 8047R