TITLE: LAMP/RT-LAMP COVID positive control
AUTHORS: Anibal Arce Medina, Isaac Núñez, Tamara Matute, Fernan Federici

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains how to prepare dsDNA and ssRNA target positive control for LAMP and RT-LAMP reactions. Particularly it describes the pipeline for SARS-Cov-2 Nucleocapsid (N) gene positive control which is included in the ReClone collection. The general procedure involves amplifying the gene of interest with primers which add T7 promoter and T7 terminator to its sequence followed by T7 in vitro transcription to obtain the ssRNA.  </div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/c3rehefe.jpg" /></div><div class = "text-block"><span style = "font-weight:bold;">Sequence elaboration:</span></div><div class = "text-block"><span>Sequence </span><a href="https://benchling.com/s/seq-75ss3ORzeIIdAdEoeXd8" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">OrfN_5</span></a><span> was designed and synthesized based on IDT SARS-CoV-2 positive control plasmid (</span><a href="https://benchling.com/s/seq-p10uFkf1oL5q76J5ym96" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">CAT#10006625_2019</span></a><span>).</span></div></div>

[STEPS]
?. [N gene Amplification]
Amplify SARS-Cov-2 Nucleocapsid (N) gene fragment dsDNA from ReClone or IDT positive control plasmid with the ultramers indicated in Table 1.The amplification can be carried out using any standard PCR reaction formulation (e.g. Phusion DNA polymerase with HF buffer), adding  to  of the plasmid template and of each primer at 10 ?M concentration and following this thermocycler program:Initial denaturation at for Followed by 35 cycles of: per  per      extension at  per End by a final extension at  per
0.01 ng
1 ng
1 µl
98 °C
98 °C
57 °C
72 °C
72 °C
?. [dsDNA positive control]
dsDNA purification Perform PCR reaction purification with any commercially available kit. We use Promega Wizard® SV Gel and PCR Clean-Up System
Typical yields are around   at 30 - 100 ng/?L.
?. [ssRNA positive control]
In vitro transcription preparationIn vitro transcription has to be done from previously prepared dsDNA. We do it using HiScribe™ (NEB E2040S) system plus PPI (NEB M0361S) and RNasin® (Promega N2111):
?. [ssRNA positive control]
Treat the reaction with DNAse I (NEB M0303S) by diluting the reaction with  H20 (molecular grade) and  10X buffer DNAse I.
70 µl
10 µl
?. [ssRNA positive control]
Incubate reaction at
37 °C
?. [ssRNA positive control]
Incubated at  per .
37 °C
Heat inactivation of DNAse is not recommended because it can harm RNA.
?. [ssRNA positive control]
Perform RNA purification with any available kit. We use Qiagen RNeasy Kit.
Typical yields are around  at 1000ng/?L.
?. [Copy number computation and dilutions]
Compute the sample copy number concentration [copies/?L] by replacing the length [bp] (Table 3)  and mass concentration [ng/?L] (C_{mass}) in the next mathematical expressions :(1.a) \text{Z}\frac{g}{\operatorname{mol} d s D N A}=\text { length } d s D N A[b p] * 617.96 \frac{g}{m o l * b p}+36.04 \frac{g}{m o l}(1.b)  \text{Z}\frac{g}{\operatorname{mol} ssR N A}=\text { length } s s R N A[b p] * 321.47 \frac{g}{m o l * b p}+18.02 \frac{g}{m o l}(2) \text{Y}\frac{\text { copies }}{n g}=\left(\text{Z}\frac{g}{\text { mol } ss R N A} * \frac{10^{9} n g}{g}\right)^{-1} * 6,02210^{23} \frac{\text { copies }}{\operatorname{mol} d s D N A}(3) \text{X}\frac{\text { copies }}{\mu L}=C_{mass}\frac{n g}{\mu L} * \text{Y}\frac{\text { copies }}{n g}
use  for dsDNA and   fo for ssRNA. \text{eq. 1.a}\text{eq. 1.b}
Typilcall amounts are:dsDNA: 108 - 109 copies/ng ssRNA: 109 - 1010 copies/ng
?. [Copy number computation and dilutions]
Make serial dilutions with molecular grade H2O to reach the target \frac{\text { copies }}{\mu L}  concentration. As positive control we typically use   of sample at \text {10.000 }\frac{\text { copies }}{\mu L}
1 µl
You can store aliquots of dsDNA samples at targets concentrations at   or  .Freezing and thawing cycles may affect the concentration then use each aliquot just one time.
?. [Copy number computation and dilutions]
In order to compute the sample copy number concentration, it's necessary to measure its mass concentration in a nano-spectrophotometer, plate reader, or estimate it by gel electrophoresis imaging.
Repeat steps 8 to 10 for ssRNA each time you perform experimentation because samples and aliquots concentration is not stable even though keeping them frozen.
?. [dsDNA positive control]
Store sample under
-20 °C
In case you are not making RT-LAMP positive control go to step 10.
?. [ssRNA positive control]
Perform aliquots and store the samples at
-80 °C