Editing Cleancap mRNA Invitro Transcription (section)

=== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2, Digestion plasmid DNA with <s>PacI, or Xho1 or</s> MluI or XbaI</span> ===

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PacI digested template has many large RNA or DNA bands,</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">For T7-VEE, PacI&nbsp; and XhoI produces 3´overhang, so they are not good for linearization, if PacI or Xho1 is to be used, DNA template must be blunted by&nbsp; adding DNA Polymerase I Large (Klenow) Fragment at a concentration of 5u/μg and incubate the reaction&nbsp;mixture for 15 minutes at 22°C.&nbsp;&nbsp;</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''For &nbsp;digestion of 36 ug of T7-VEE plasmid in 200&nbsp;μL'''</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">36 μL T7-VEE Plasmid (1 μg/μL)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">20 μL CutSmart Buffer (10X)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">139 μL Sterile distilled water</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">5 μL MluI-HF (10,000 units/mL, NEB)</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Incubate digestion mixtures at 37°C for 3 hours.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Add the same volume (200 μL) of phenol/chloroform/isoamyl alcohol (25:24:1) to each eppendorf tube containing the digestion mixture.<br/>Vortex the eppendorf tubes for 60 seconds. Centrifuge at high speed (14,000 rpm) for 5 minutes.<br/>Transfer each aqueous phase (top layer) into new eppendorf tubes.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Note: T7-VEE plasmid is a large sized DNA and may be trapped in the interphase layer with the proteins. When the interphase is sticky (DNA is trapped), collect the interphase layer along with the aqueous layer.<br/>To remove the excess phenol, add an equal volume (200 μL) of chloroform/isoamyl alcohol (24:1) to each eppendorf tubes.<br/>Vortex the eppendorf tubes for 60 seconds and centrifuge at high speed (14,000 rpm) for 5 minutes.<br/>Transfer each aqueous phase (top layer) into new eppendorf tubes.<br/>Add 1/10 volume (20 μL) of 3 M sodium acetate, pH 5.2 (this can be found in the RiboMAX Large Scale RNA Production kit) and 2.5 X volume (500 μL) of 100% ethanol to each eppendorf tubes.<br/>Vortex and place at -80°C for 5 minutes or store at -20°C until use.<br/>Note: If there are no plans to proceed to RNA synthesis on the same day, stop at this step. DNA will be stable for several months at -20°C.<br/>Centrifuge at high speed (14,000 rpm) for 10-15 minutes at 4°C to pellet the DNA plasmid. Remove the ethanol carefully with a pipet tip, being careful not to dislodge the DNA pellet.<br/>Gently add 1 mL 70% ethanol to wash the DNA pellet. Centrifuge at high speed at 4°C for 1 minute. Remove the ethanol carefully with a pipet tip, being careful not to dislodge the RNA pellet.<br/>Centrifuge shortly, and carefully remove any visible residual ethanol with a P20 pipette. Drying the DNA pellet is not necessary. In general, approximately 20% of plasmid may be lost during purification.<br/>Resuspend the DNA pellet in a small volume (15~20 μL, depending on starting amount) of DNase & RNase-free water before measuring the concentration.<br/>Measure the DNA concentration of each linearized DNA plasmid. Adjust the concentration to 1 μg/μL.</span>





<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">'''mRNA analysis by agarose'''</span>

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
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| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Lane</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Desc</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Note</span>
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| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">1</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PacI digested plasmid, large scale</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">need to remove DNA using DNase I</span>
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| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">2</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PacI digested plasmid,&nbsp;small scale</span><br/>
| <br/>
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| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">3</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Xho1 digested plasmid,&nbsp;small scale</span>
| <br/>
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| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">4</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR template,&nbsp;small scale</span>
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| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">5</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">DNA ladder, NEB, 1 Kb</span>
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| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">6</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR template 1, precipitate with isopropanol + glycogen</span><br/>
| <br/>
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| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">7</span>
| <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">PCR template 2, precipitate with isopropanol</span><br/>
| <br/>
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<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">[[File:ShenDong mRNA QC 12 14 2020 long.JPG|RTENOTITLE]]</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">mRNA made from 6 + 7 combined, 4 tubes of 40 ul reaction from template 6,&nbsp;4 tubes of 40 ul reaction from template 7, purified using Qiagen RNeasy miniprep kit</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">[[File:MRNA2.jpg|RTENOTITLE]]</span>



<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">CleanCap is used for easy one -step reaction, while enzymatic capping method requires three-step reaction but it minimizes the IFN responses by combination with phosphatase treatment</span>