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16S and ITS sequencing - Progress/Plates and Assigned MIDs

16S and ITS sequencing - Progress/Plates and Assigned MIDs

Steps for completion of 16S/ITS sequencing of Alfalfa samples. Checked boxes indicate task complete.

#PCR currently on hold due to issues with PCR plates. PCR plates have entire well evaporation throughout the plates once plates are refrigerated, even with 8 strip bubble caps. Speaking with manufacturer for resolution…should be responding with possible solutions 5/10.

**Steps are for internal samples only.

Quantify and normalize _END plates 1-8
Quantify and normalize _GBS plates 1-11
Quantify and normalize _EPI plates 1-8
PCR _END plates 1-8
Clean _END plates 1-8
qPCR _END plates 1-8 (2 samples from each plate with standards)
PCR _GBS plates 1-11
Clean _GBS plates 1-11
qPCR _GBS plates 1-11 (2 samples from each plate with standards)
PCR _EPI plates 1-9
Clean _EPI plates 1-9
qPCR _EPI plates 1-9 (2 samples from each plate with standards)
Quantify via absorbance for pooling
Normalize PCR
Pool products and check pool on qPCR
Prepare final pool for NovaSeq based on qPCR results

16S/ITS Plates

16S MID

ITS MID

ALF_PLT1_2020_END

long16S0A1

longITS0A1

long16S0B1

longITS0B1

ALF_PLT2_2020_END

long16S0C1

 longITS0C1

 long16S0D1

longITS0D1

ALF_PLT3_2020_END

long16S0E1

longITS0E1

long16S0F1

longITS0F1

ALF_PLT4_2020_END

long16S0G1

longITS0G1

 long16S0H1

 longITS0H1

ALF_PLT5_2020_END

 long16S0A2

 longITS0A2

 long16S0B2

 longITS0B2

ALF_PLT6_2020_END

 long16S0C2

 longITS0C2

 long16S0D2

 longITS0D2

ALF_PLT7_2020_END

 long16S0E2

 longITS0E2

 long16S0F2

 longITS0F2

ALF_PLT8_2020_END

 long16S0G2

 longITS0G2

 long16S0H2

 longITS0H2

ALF_PLT1_2020_EPI

 long16S0A3

 longITS0A3

 long16S0B3

 longITS0B3

ALF_PLT2_2020_EPI

 long16S0C3

 longITS0C3

 long16S0D3

 longITS0D3

ALF_PLT3_2020_EPI

 long16S0E3

 longITS0E3

 long16S0F3

 longITS0F3

ALF_PLT4_2020_EPI

 long16S0G3

 longITS0G3

 long16S0H3

 longITS0H3

ALF_PLT5_2020_EPI

 long16S0A4

 longITS0A4

 long16S0B4

 longITS0B4

ALF_PLT6_2020_EPI

 long16S0C4

 longITS0C4

 long16S0D4

 longITS0D4

ALF_PLT7_2020_EPI

 long16S0E4

 longITS0E4

 long16S0F4

 longITS0F4

ALF_PLT8_2020_EPI

 long16S0G4

 longITS0G4

 long16S0H4

 longITS0H4

ALF_PLT9_2020_EPI

 long16S0G7

 longITS0G7

 long16S0H7

 longITS0H7

ALF_PLT1_2020_SOIL

 long16S0A8

 longITS0A8

 long16S0B8

 longITS0B8

ALF_PLT1_2017_GBS

 long16S0A5

 longITS0A5

 long16S0B5

 longITS0B5

ALF_PLT2_2017_GBS

 long16S0C5

 longITS0C5

 long16S0D5

 longITS0D5

ALF_PLT3_2017_GBS

 long16S0E5

 longITS0E5

 long16S0F5

 longITS0F5

ALF_PLT4_2017_GBS

 long16S0G5

 longITS0G5

 long16S0H5

 longITS0H5

ALF_PLT5_2017_GBS

 long16S0A6

 longITS0A6

 long16S0B6

 longITS0B6

ALF_PLT6_2017_GBS

 long16S0C6

 longITS0C6

 long16S0D6

 longITS0D6

ALF_PLT7_2017_GBS

 long16S0E6

 longITS0E6

 long16S0F6

 longITS0F6

ALF_PLT8_2017_GBS

 long16S0G6

 longITS0G6

 long16S0H6

 longITS0H6

ALF_PLT9_2017_GBS

 long16S0A7

 longITS0A7

 long16S0B7

 longITS0B7

ALF_PLT10_2017_GBS

 long16S0C7

 longITS0C7

 long16S0D7

 longITS0D7

ALF_PLT11_2017_GBS

 long16S0E7

 longITS0E7

 long16S0F7

 longITS0F7

Steps for completion of 16S/ITS sequencing of Alfalfa Tennessee samples. Checked boxes indicate task complete.

Transfer 30uL from the submitted plate to a new clear plate (check plates for cracks before use due to recent broken plates sent from manufacturer) and add 6uL of coligos. Label plate with GTL designated name and seal with a foil seal.
Vortex and spin down plate after coligo addition and quantify on the synergy HTX.
Normalize plates to 10ng/uL on Hamilton Nimbus. Label plates with GTL given name_Norm, seal with foil, and place in -20.
PCR
Clean-Up
qPCR

 

Alfalfa Plates from Tennessee (Possible separate Novaseq run, MIDs to be determined)

16S/ITS Plates

16S MID

ITS MID

ALF_PLT1_TN

 long16S0G7

 longITS0G7

 long16S0H7

 longITS0H7

ALF_PLT2_TN

long16S0A8

longITS0A8

long16S0B8

longITS0B8

ALF_PLT5_TN

long16S0C8

longITS0C8

long16S0D8

longITS0D8

ALF_PLT6_TN

long16S0E8

longITS0E8

long16S0F8

longITS0F8

ALF_PLT9_TN

long16S0G8

longITS0G8

long16S0H8

longITS0H8

ALF_PLT10_TN

long16S0A9

longITS0A9

long16S0B9

longITS0B9

ALF_PLT11_TN

long16S0C9

longITS0C9

long16S0D9

longITS0D9

ALF_PLT12_TN

long16S0E9

longITS0E9

long16S0F9

longITS0F9

ALF_PLT15_TN

long16S0G9

longITS0G9

long16S0H9

longITS0H9

ALF_PLT17_TN

long16S0A10

longITS0A10

long16S0B10

longITS0B10

ALF_PLT18_TN

long16S0C10

longITS0C10

long16S0D10

longITS0D10

ALF_PLTA_TN

long16S0E10

longITS0E10

long16S0F10

longITS0F10

ALF_PLTB_TN

long16S0G10

longITS0G10

long16S0H10

longITS0H10

ALF_PLTC_TN

long16S0A11

longITS0A11

long16S0B11

longITS0B11

ALF_PLTD_TN

long16S0C11

longITS0C11

long16S0D11

longITS0D11