Versions Compared

Version Old Version 7 New Version 8
Changes made by

Shannon Harris

Shannon Harris

Saved on

Key

  • This line was added.
  • This line was removed.
  • Formatting was changed.

Gregg Randolph Linda van Diepen Alex Buerkle

...

  1. Pull aliquots of some high read count and low read count samples. (Pending new low read samples).

  2. Quantify all samples not including blanks or mock community.

  3. For previously low yielding samples, repeat at typical template amount, and at 2x, and 4x volume added to the reaction (in column 2,3,4, and 6, 7, 8). 6x8 samples in low yield, 8 in high yield, 8 of mock community, 8 of blank (ISD only). 72 reactions with different templates. See template below for clarification.

  4. Template layout is replicated/duplicated, with different barcodes for duplicates.

  5. 1 set of 36 products will not be adjusted for pooling.

  6. 1 set of 36 will be adjusted for pooling per qPCR results.

Start Reaction Plate Setup:

...

 

1

2

3

4

5

6

7

8

9

10

11

12

A

SAG192203

Tube 127

2ul SAG_S30P2R

PLT2: G6

4ul SAG_S30P2R

PLT2: G6

8ul SAG_S30P2R

PLT2: G6

mock community

2ul SAG192199

Tube 123

4ul SAG192199

Tube 123

8ul SAG192199

Tube 123

 blank with ISD only

NTC

NTC

NTC

B

SAG191508

Tube 73

2ul SAG_S38P1R

PLT2: B9

4ul SAG_S38P1R

PLT2: B9

8ul SAG_S38P1R

PLT2: B9

mock community

2ul SAG192077

Tube 96

4ul SAG192077

Tube 96

8ul SAG192077

Tube 96

blank with ISD only

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

SAG_S16P3R

PLT1: G3

2ul SAG_S15P4R

PLT2: B2

4ul SAG_S15P4R

PLT2: B2

8ul SAG_S15P4R

PLT2: B2

mock community

2ul SAG190271

Tube 258

4ul SAG190271

Tube 258

8ul SAG190271

Tube 258

  blank with ISD only

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

SAG_S44P2R

PLT1: F7

2ul SAG190005

Tube 33

4ul SAG190005

Tube 33

8ul SAG190005

Tube 33

mock community

2ul SAG190238

Tube 255

4ul SAG190238

Tube 255

8ul SAG190238

Tube 255

  blank with ISD only

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

SAG192203

Tube 127

2ul SAG_S30P2R

PLT2: G6

4ul SAG_S30P2R

PLT2: G6

8ul SAG_S30P2R

PLT2: G6

mock community

2ul SAG192199

Tube 123

4ul SAG192199

Tube 123

8ul SAG192199

Tube 123

 blank with ISD only

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

SAG191508

Tube 73

2ul SAG_S38P1R

PLT2: B9

4ul SAG_S38P1R

PLT2: B9

8ul SAG_S38P1R

PLT2: B9

mock community

2ul SAG192077

Tube 96

4ul SAG192077

Tube 96

8ul SAG192077

Tube 96

blank with ISD only

2 pM Std

2 pM Std

2 pM Std

G

SAG_S16P3R

PLT1: G3

2ul SAG_S15P4R

PLT2: B2

4ul SAG_S15P4R

PLT2: B2

8ul SAG_S15P4R

PLT2: B2

mock community

2ul SAG190271

Tube 258

4ul SAG190271

Tube 258

8ul SAG190271

Tube 258

  blank with ISD only

20 pM Std

20 pM Std

20 pM Std

H

SAG_S44P2R

PLT1: F7

2ul SAG190005

Tube 33

4ul SAG190005

Tube 33

8ul SAG190005

Tube 33

mock community

2ul SAG190238

Tube 255

4ul SAG190238

Tube 255

8ul SAG190238

Tube 255

  blank with ISD only

 

 

 

Results:

View file
nameLRII_16S_B_R.csv

Pool all samples:

Blue samples (top half of plate) will be pooled at 1* ul per sample.

Red samples (bottom half) will either just be pooled per qPCR numbers or concentrated via SpeedVac, reconstituted to a higher concentration, and pooled by qPCR results. (Pending method instructions).

qPCR blue/red pools:

  • Make 1:1000 dilutions of blue and red pools by adding 1 ul to 999 ul TE in a 1.5mL tubes.

  • Run each pool in triplicate.

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

...