Last updated: 2022-10-06

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Aim

The aim of this test is to assess the impact of retraction speed of Pintool to its stamping accuracy with help from Adam (PE). We have also tested if the pin retraction height made any difference.

Adam had acknowledged the issue with faulty pins discovered in the previous tests and the Pintool adapter stability issue. The problem with Pintool adapter stability might be the slight Z direction drift which caused it to not latching properly.

We also agreed on I should bve testing the pre-wetting of pintool to see if a thinning agent (in this case, water) will make the pintool liquid handling better.

Another test that we wanted to do is the wash station. We did see the 384 head can also pick up the pintool. So maybe the existing wash station (with pumps) in VCFG can be used for Pintool wash.

Researcher

ML

Readout

Absorbance of tartrazine at 410 on Cytation 5 plate reader

Required R packages

data.table, DT, platetools, reshape2, tidyverse, patchwork

# load the required packages
library(data.table)
library(DT)
library(platetools)
library(reshape2)
library(readxl)
library(tidyverse)
library(patchwork)
library(here)
library(htmltools)


# check if here() correctly identified the directory
here::here()
[1] "/home/mli/ML_Mac-Seq/Pintool_dev/220920_test"
# set the file prefix

prefix <- "ML_Mac-Seq_Pintool_dev"

Screen details

Screen date (yyyy-mm-dd)

2022-09-20

Tartrazine concentration

Tartrazine stock at 12.5 ug/ul Source plate made @ tartrazine 10 ug/ul with 1X PBS

Standard curve

Standard curve of tartrazine absorbance (Abs) was constructed by triplicate serial dilution

Plate layout



Technical details

Plate reader Abs signal saturation occurs between 0.08 to 0.16 ug/ul tartrazine.

Fresh Standard curve plate was prepared. A new PBS plate was set to wash station 1 so we can measure the residual dye on the pintool after delivery.

3 retraction speed tested in Aspiration (A) (10mm/sec, 30mm/sec and 200mm/sec) 1 retraction speed tested in Dispense (D) (200mm/sec)

Clearing height tested: default to 10mm

Due the number of conditions tested, the wash plate was reused so we need to compare the delta of each reading.

Data cleaning

Formatting and Preprocessing

The raw data was read into R Studio.

Data processing

Pintool Delivery Quality

Pintool Consistency Heatmaps


Comments: I would like to look at the standard curve plate, and target plate after each transfer

Abs raw values for STD and Target plates


Abs Z score per plate


Abs Delta over each previous transfer

Target Plates


Wash Plates

Pintool Accuracy

Standard Curve


Comments: Now that I know the consistency is not ideal, I want to know just how accurate the pintool is in delivery the labelled 100nl volume. In theory, a 100nl of my stock 10ug/ul tartrazine trasnferred into 50ul PBS should give a final 0.02 ug/ul (i.e. 1ug dye increament). Using the standard curve constructed, I can get a portion of it where Abs readings and dye concentrations approximated a linear relationship (unfortunately the next dye concentration 0.16 ug/ul had an Abs value beyond detection limit). In this roughly linear section, tartrazine concentration covered is ranging 0.002 ug/ul to 0.08 ug/ul, covering the final concentration for at least 4 transfers in this test. I can use this to estimate the dye (hence real volume) transferred by each pin at each transfer, see how far off is that compared to 100nl



Call:
lm(formula = Conc ~ Abs, data = Standard.curve)

Coefficients:
(Intercept)          Abs  
  -0.003763     0.018658


Comments: Roughly using a linear approximation: Concentration = (0.019xAbs - 0.003) ug/ul.The volume change by trasnferring a couple rounds of 100nl into 50ul well is less than 1%. So we can have the relationship of Volume = (0.095xAbs - 0.015)x1000 nl

Convert Abs Delta to Volume

Comments:


 

Analysed by Mark Li

Victorian Centre for Functional Genomics

 


sessionInfo()
R version 4.2.0 (2022-04-22)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)

Matrix products: default
BLAS:   /usr/lib64/libblas.so.3.4.2
LAPACK: /usr/lib64/liblapack.so.3.4.2

locale:
 [1] LC_CTYPE=en_AU.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_AU.UTF-8        LC_COLLATE=en_AU.UTF-8    
 [5] LC_MONETARY=en_AU.UTF-8    LC_MESSAGES=en_AU.UTF-8   
 [7] LC_PAPER=en_AU.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_AU.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
 [1] htmltools_0.5.3   here_1.0.1        patchwork_1.1.2   forcats_0.5.2    
 [5] stringr_1.4.1     dplyr_1.0.10      purrr_0.3.4       readr_2.1.2      
 [9] tidyr_1.2.0       tibble_3.1.8      ggplot2_3.3.6     tidyverse_1.3.2  
[13] readxl_1.4.1      reshape2_1.4.4    platetools_0.1.5  DT_0.24          
[17] data.table_1.14.2 workflowr_1.7.0  

loaded via a namespace (and not attached):
 [1] nlme_3.1-159        fs_1.5.2            lubridate_1.8.0    
 [4] bit64_4.0.5         RColorBrewer_1.1-3  httr_1.4.4         
 [7] rprojroot_2.0.3     tools_4.2.0         backports_1.4.1    
[10] bslib_0.4.0         utf8_1.2.2          R6_2.5.1           
[13] DBI_1.1.3           colorspace_2.0-3    withr_2.5.0        
[16] tidyselect_1.1.2    processx_3.7.0      bit_4.0.4          
[19] compiler_4.2.0      git2r_0.30.1        cli_3.3.0          
[22] rvest_1.0.3         formatR_1.12        xml2_1.3.3         
[25] labeling_0.4.2      sass_0.4.2          scales_1.2.1       
[28] callr_3.7.2         digest_0.6.29       rmarkdown_2.16     
[31] pkgconfig_2.0.3     highr_0.9           dbplyr_2.2.1       
[34] fastmap_1.1.0       htmlwidgets_1.5.4   rlang_1.0.5        
[37] rstudioapi_0.14     farver_2.1.1        jquerylib_0.1.4    
[40] generics_0.1.3      jsonlite_1.8.0      crosstalk_1.2.0    
[43] vroom_1.5.7         googlesheets4_1.0.1 magrittr_2.0.3     
[46] Rcpp_1.0.9          munsell_0.5.0       fansi_1.0.3        
[49] lifecycle_1.0.1     stringi_1.7.8       whisker_0.4        
[52] yaml_2.3.5          plyr_1.8.7          grid_4.2.0         
[55] parallel_4.2.0      promises_1.2.0.1    crayon_1.5.1       
[58] lattice_0.20-45     splines_4.2.0       haven_2.5.1        
[61] hms_1.1.2           knitr_1.40          ps_1.7.1           
[64] pillar_1.8.1        reprex_2.0.2        glue_1.6.2         
[67] evaluate_0.16       getPass_0.2-2       modelr_0.1.9       
[70] vctrs_0.4.1         tzdb_0.3.0          httpuv_1.6.5       
[73] cellranger_1.1.0    gtable_0.3.1        assertthat_0.2.1   
[76] cachem_1.0.6        xfun_0.32           mime_0.12          
[79] broom_1.0.1         later_1.3.0         googledrive_2.0.0  
[82] gargle_1.2.0        ellipsis_0.3.2