TITLE: Preparation of 5M Guanidine thiocyanate L6 Inactivation Buffer
AUTHORS: Nicola O'Reilly, Svend Kjaer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purpose of examination / Clinical relevance</span></div><div class = "text-block">At the end of 2019, several pneumonia cases were reported in Wuhan, China and the pathogen was confirmed as a new viral strain. World Health organization has named the newly identified coronavirus as 2019-nCoV, also known as COVID19. The disease developed into a dangerous pandemic, posing major challenges to the NHS. Although more research is necessary to better understand the virus, in response to the emergency, simple and rapid testing is essential to identify the virus in infected individuals. This will aid the implementation of efficient interventions to contain the spread, and distinguish healthcare workers who have been infected, and are required to self-isolate, from those showing similar symptoms but which are not 2019-nCoV associated. The latter category may continue to work, alleviating stress on hard-pressed healthcare resources. 2019-nCoV is an RNA virus, and the diagnostic tests detect viral RNA in swabs from patient airways using a reverse transcriptase PCR assay. Samples are submitted to HSL, an accredited reporting laboratory, and transferred to the Crick for testing.</div><div class = "text-block"><span style = "font-weight:bold;">Principles of Examination</span></div><div class = "text-block"><span>This procedure involves the preparation of L6 5M Guanidine thiocyanate virus inactivation buffer for the </span><a href="https://www.protocols.io/private/E84AE35681AE11EABF5D0242AC110002" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">viral inactivation</span></a><span> protocol.</span></div></div>

[STEPS]
?. [Preparation of L6 Lysis Buffer]
Please select between the recipes for 1 L, 2 L, 4.3 L and 5.4 L L6 5 M Guanidine thiocyanate Inactivation buffer.
Upon contact with acids, GuSCN can produce a toxic gas (HCN). As a precaution, GuSCN-containing buffers are prepared in a fume hood.
?. [Preparation of L6 Lysis Buffer]
Weigh out  and add to a 1 L beaker.
[GuSCN]
?. [Preparation of L6 Lysis Buffer]
Measure out  and add to beaker.
[0.1 M Tris HCl]
?. [Preparation of L6 Lysis Buffer]
Measure out ,  and add to beaker. Stir and heat to  if required.
[0.2 M EDTA]
60 °C
?. [Preparation of L6 Lysis Buffer]
Once GuSCN is dissolved, add  to beaker and stir well.
[Triton X-100]
?. [Preparation of L6 Lysis Buffer]
Vacuum filter into sterile bottles.
?. [Preparation of L6 Lysis Buffer]
Label with batch and date.
?. [Preparation of L6 Lysis Buffer]
Store L6 inactivation buffer in dark at solution at  ( – ).
0 Room temperature
15 °C
25 °C
?. [Preparation of L6 Lysis Buffer]
Weigh out  and add to a 2 L beaker.
[GuSCN]
?. [Preparation of L6 Lysis Buffer]
Measure out  and add to beaker.
[0.1 M Tris HCl]
?. [Preparation of L6 Lysis Buffer]
Measure out ,  and add to beaker and heat to  if required.
[0.2 M EDTA]
60 °C
?. [Preparation of L6 Lysis Buffer]
Once GuSCN is dissolved, add  to beaker and stirr well.
[Triton X-100]
?. [Preparation of L6 Lysis Buffer]
Vacuum filter into sterile bottles.
?. [Preparation of L6 Lysis Buffer]
Label with batch and date.
?. [Preparation of L6 Lysis Buffer]
Store L6 inactivation buffer in dark at solution at  ( – ).
0 Room temperature
15 °C
25 °C
?. [Preparation of L6 Lysis Buffer]
Weigh out  in aliquots using a 3 L beaker and carefully add to 5 L beaker whilst stirring and heating to . Rinse 3 L beaker with . Stir manually with sterile 10 ml pipette if required.
[GuSCN]
60 °C
[0.1 M Tris HCl]
?. [Preparation of L6 Lysis Buffer]
Measure out  and add to 5 L beaker.
[0.1 M Tris HCl]
?. [Preparation of L6 Lysis Buffer]
Vacuum filter into sterile bottles.
?. [Preparation of L6 Lysis Buffer]
Label with batch and date.
?. [Preparation of L6 Lysis Buffer]
Store L6 inactivation buffer in dark at solution at  ( – ).
0 Room temperature
15 °C
25 °C
?. [Preparation of L6 Lysis Buffer]
Measure out ,  and add to beaker.
[0.2 M EDTA]
?. [Following Preparation of L6 Lysis Buffer]
Within a tissue culture hood cupboard, aliquot  into 2 ml tubes and replace lids.
[5 M guanidine thiocyanate]
?. [Following Preparation of L6 Lysis Buffer]
Label with batch and date and store in dark at .
0 Room temperature
?. [Following Preparation of L6 Lysis Buffer]
After the aliquoting is complete, change gloves.
?. [Following Preparation of L6 Lysis Buffer]
Place tubes in a box clearly marked 5 M guanidine thiocyanate L6 Lysis buffer.
?. [Following Preparation of L6 Lysis Buffer]
Clean hood, dispose of waste and switch off tissue culture hood.
5 M Guanidine thiocyanate is stable at   for at least three weeks.(Boom et al, J Clin Microbiol 1990. 28, 3, 485-503 . Commercial stocks have shelf life of 18 months)
?. [Following Preparation of L6 Lysis Buffer]
Within a tissue culture hood cupboard, aliquot  into 2 ml tubes and replace lids.
[5 M guanidine thiocyanate]
?. [Following Preparation of L6 Lysis Buffer]
Label with batch and date and store in dark at .
0 Room temperature
?. [Following Preparation of L6 Lysis Buffer]
After the aliquoting is complete, change gloves.
?. [Following Preparation of L6 Lysis Buffer]
Place tubes in a box clearly marked 5 M guanidine thiocyanate L6 Lysis buffer.
?. [Following Preparation of L6 Lysis Buffer]
Clean hood, dispose of waste and switch off tissue culture hood.
5 M Guanidine thiocyanate is stable at   for at least three weeks.(Boom et al, J Clin Microbiol 1990. 28, 3, 485-503 . Commercial stocks have shelf life of 18 months)
?. [Following Preparation of L6 Lysis Buffer]
Within a tissue culture hood cupboard, aliquot  into 2 ml tubes and replace lids.
[5 M guanidine thiocyanate]
?. [Following Preparation of L6 Lysis Buffer]
Label with batch and date and store in dark at .
0 Room temperature
?. [Following Preparation of L6 Lysis Buffer]
After the aliquoting is complete, change gloves.
?. [Following Preparation of L6 Lysis Buffer]
Place tubes in a box clearly marked 5 M guanidine thiocyanate L6 Lysis buffer.
?. [Following Preparation of L6 Lysis Buffer]
Clean hood, dispose of waste and switch off tissue culture hood.
5 M Guanidine thiocyanate is stable at   for at least three weeks.(Boom et al, J Clin Microbiol 1990. 28, 3, 485-503 . Commercial stocks have shelf life of 18 months)
?. [Preparation of L6 Lysis Buffer]
Measure out  and add to 5 L beaker.
[0.1 M Tris HCl]
?. [Preparation of L6 Lysis Buffer]
Weigh out  in aliquots using a 3 L beaker and carefully add to 5 L beaker whilst stirring and heating to . Rinse 3 L beaker with  and add to 5 L beaker. Stir manually with sterile 10 ml pipette if required.
[GuSCN]
60 °C
[0.1 M Tris HCl]
?. [Preparation of L6 Lysis Buffer]
Measure out ,  and add to bottle.
[0.2 M EDTA]
?. [Preparation of L6 Lysis Buffer]
Add  to bottle. Swirl well to mix.
[Triton X-100]
?. [Preparation of L6 Lysis Buffer]
Vacuum filter into sterile bottles.
?. [Preparation of L6 Lysis Buffer]
Label with batch and date.
?. [Preparation of L6 Lysis Buffer]
Store L6 inactivation buffer in dark at solution at  ( – ).
0 Room temperature
15 °C
25 °C
?. [Following Preparation of L6 Lysis Buffer]
Within a tissue culture hood cupboard, aliquot  into 2 ml tubes and replace lids.
[5 M guanidine thiocyanate]
?. [Following Preparation of L6 Lysis Buffer]
Label with batch and date and store in dark at .
0 Room temperature
?. [Following Preparation of L6 Lysis Buffer]
After the aliquoting is complete, change gloves.
?. [Following Preparation of L6 Lysis Buffer]
Place tubes in a box clearly marked 5 M guanidine thiocyanate L6 Lysis buffer.
?. [Following Preparation of L6 Lysis Buffer]
Clean hood, dispose of waste and switch off tissue culture hood.
5 M Guanidine thiocyanate is stable at   for at least three weeks.(Boom et al, J Clin Microbiol 1990. 28, 3, 485-503 . Commercial stocks have shelf life of 18 months)
?. [Preparation of L6 Lysis Buffer]
Once GuSCN is dissolved, add  to beaker. Stir well to mix.
[Triton X-100]
?. [Preparation of L6 Lysis Buffer]
Once GuSCN is dissolved, transfer to 5 L single use tissue culture bottle.