Current Protocols

 

 

 

Rothamsted Research is one if eight institutes sponsored by the biotechnology and biological sciences research council

 

 

 

 

 

 

 

 

 

 

 

 

Our current protocols are outlined below. Many of our methods are still under development and will be posted here as they develop into standard operating procedures. Please check back regularly to see our latest procedures

 

  

 

  

 

 

 

 

Current Protocol for Growth of Arabidopsis

SOP for Germination of Arabidopsis

SOP for Transferral to Soil

SOP for Harvesting

SOP for the Preparation of Plant Material for Analysis

 

 

 
Current Protocol for NMR Sample Preparation

SOP for Preparation of NMR extracts

 

(This method gives a polar extract and is currently used for Arabidopsis, wheat and potato samples)

 

 

 

Parameter Sets for NMR Data Collection

Parameters for standard “tube” NMR analysis of samples in D2O/CD3OD using the400 MHz NMR.

 

Instrument Parameters

Instrument

BrukerBiospin, Avance 400

Probe

5mm broad band multinuclear probe (BBO Z8248/0048)

Proton Frequency

400.1299232 MHz

Sample Introducion System

BACS

Automation Control Software

ICON-NMR

NMR acquisition Software

Xwin-NMR 9 (v3.5)

 

 

Acquisition Parameters

Parameter Set

Watersup.roth

Pulse Sequence

zgpr (low power presaturation pulse followed by high power 90˚ pulse)

Presturation pulse

50.0 dB* for 5 s

90˚ pulse

-6 dB* for 7.9 µs

Sweep width

12.1084 ppm

Time Domain size

32k data points

Temperature

300 K

Irradiation Frequency/

transmitter offset

4.7ppm

Number of Scans

1024**

Lock

Automated locking on D2O

Sample Shimming

tune xyz (all under automation)

Sample Spinning

20 Hz

Receiver Gain

Optimised for each sample by the  automation program au_zg (using command RGA)

 

 

Spectral Processing Parameters(using X-WinNMR)

Spectral width

32k real points (no zero filling)

Window function

EM (exponential multiplication)

Line Broadening (for EM)

0.5 Hz

Baseline Correction

Automated using 5th order polynomial

Spectrum referencing

Referenced to TSP-D4 at 0.00 ppm

 

 

Preparation for transfer to Spectra Base (using Amix)

Noise level definition

Calculated from noise region

Noise region

10.1 to 10.0 ppm

Noise factor

10

Negative Peaks

Removed

* pulse attenuations are spectrometer/probe/sample-type specific.

** value subject to change between experiments (i.e. sets of samples)


 

Parameters for standard “tube” NMR analysis of samples in D2O/CD3OD using the 600MHz NMR.

 

Instrument Parameters

Instrument

BrukerBiospin, Avance 600

Probe

5mm selective inverse probe (SEI Z28381/0012)

Proton Frequency

600.0528202 MHz

Sample Introducion System

BACS

Automation Control Software

ICON-NMR

NMR acquisition Software

Topspin (v1.3)

 

 

Acquisition Parameters

Parameter Set

Watersup.rothspin

Pulse Sequence

zgpr (low power presaturation pulse followed by high power 90˚ pulse)

Presturation pulse

56.42 dB* for 5 s

90˚ pulse

2 dB* for 11.1 µs

Sweep width

12.1084 ppm

Time Domain size

64k data points

Delay

5s

Temperature

300 K

Irradiation Frequency

4.7ppm

Number of Scans

128**

Lock

Automated locking on D2O

Sample Shimming

1D Deuterium Gradient Shim

Sample Spinning

20 Hz

Receiver Gain

Optimised for each sample by the  automation program au_zg (using command RGA)

 

 

Spectral Processing Parameters(using Topspin)

Spectral width

32k real points (no zero filling)

Window function

EM (exponential multiplication)

Line Broadening (for EM)

0.5 Hz

Baseline Correction

Automated using 2nd order polynomial

Spectrum referencing

Referenced to TSP-D4 at 0.00 ppm

QA

Half height line width of TSP peak is calculated under automation.  Spectra with TSP peaks wider than 1.6 Hz are rerun.

 

 

Preparation for transfer to Spectra Base (using Amix)

Noise level definition

Calculated from noise region

Noise region

10.1 to 10.0 ppm

Noise factor

10

Negative Peaks

Removed

 

 

* pulse attenuations are spectrometer/probe/sample-type specific.

** value subject to change between experiments (i.e. sets of samples)

 


 

Protocol for Data Reduction of the NMR spectra

 

The 1H NMR spectra are automatically reduced to ASCII files using AMIX (Analysis of MIXtures software v.3.0, Bruker Biospin). Spectra are scaled to d4 TSP and reduced to integrated regions or “buckets” of equal width (0.01 ppm) corresponding to the region of d9.0 to d-0.5. The regions between d4.90 and d4.76 are removed prior to statistical analyses eliminating any variability in suppression of the water signal. The signals corresponding to d4 methanol(d3.365- d3.285)  and d4 TSP (d0.00) are also removed at this stage. The generated ASCII file is imported into Microsoft EXCEL for the addition of labels and then imported into SIMCA-P 9.0 (Umetrics, Umea, Sweden) for multivariate analysis.

 
 
 

 

Current Protocols for GC-MS

 

Protocols for GC-MS extraction and derivatisation are under evaluation and will be posted only when a robust and reliable method has been established.

 

 
Current Protocols for Targeted Analyses

 

SOP for Anthocyanin Analysis

SOP for Carotenoid Analysis

SOP for Flavonoid Analysis

SOP for Phenylpropanoid Analysis

SOP for Polyamine Analysis