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Gregg Randolph

Gregg Randolph

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Plates: 2AMF1 and 2AMF2

 

1

2

3

4

5

6

7

8

9

10

11

12

1

A

2

23

34

44

54

63

71

92

 185

193

201

209

216

B

6

24

36

45

55

64

72

178 

186 

194

202

210

217

C

15

26

38

46

56

65

85

179 

187 

195

203

211

218

D

16

28

39

47

57

66

86

180 

188 

196

204

212

219

E

19

29

40

48

58

67

87

181 

189 

197

205

213

220

F

20

30

41

50

59

68

88

182 

190 

198

206

214

221

G

21

32

42

51

61

69

90

183 

191 

199

207

215

222

H

22

33

43

52

62

70

91

184 

192 

200

208

Blank

Blank2

Remaining 2 columns are Mock Community

PCR MasterMix

ul/rxn

Reagent

# of rxns

ul needed

3

5X Kapa HiFi Buffer

530

1590

0.45

10M dNTPs

530

238

0.3

Kapa HiFi HotStart DNA Pol

530

159

7.25

HPLC H2O

530

3843

11

Total Volume

530

5830

...

Plate

18S Primer

ITS

2AMF1

AMF01

ITS0E11

AMF02

ITS0F11

2AMF2

AMF03AMF04

ITS0G11

Run AMF on thermocycler program AMF35:

...

  • Make 1:1000 dilutions of column 1,6,10 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

 

1

2

3

4

5

6

7

8

9

10

11

12

A

 

NTC

NTC

NTC

B

 

 

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

 

 

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

 

 

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

 

 

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

 

 

2 pM Std

2 pM Std

2 pM Std

G

 

 

20 pM Std

20 pM Std

20 pM Std

H

 

 

 

 

 

  • Add 16 ul of Illumina Library Quantification MasterMix to each well:

...