Paul.xu at 15:44, 13 February 2022

2022-02-13T15:44:44Z

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== Linearization of plasmid DNA ==

Linearize 10 ug of plasmid DNA in a 100 uL restriction enzyme digestion as follows:

#Reaction Assembly, a total of 100 uL:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | Reagents
| bgcolor="#eaeaea" | Volume (uL)
|-
| bgcolor="#ffffff" | H20
| bgcolor="#ffffff" | 70
|-
| bgcolor="#eaeaea" | 10 x Buffer
| bgcolor="#eaeaea" | 10
|-
| bgcolor="#ffffff" | DNA (10 ug)
| bgcolor="#ffffff" | 10 (For 1ug/uL DNA)
|-
| bgcolor="#eaeaea" | Enzyme
| bgcolor="#eaeaea" | 10
|-
! bgcolor="#dadada" | Total
! bgcolor="#dadada" | 100
|}

#Digest 3 hours at 37° (or whatever temperature the restriction enzyme requires).
#Run out 0.5 ug (5 uL) of linearized DNA with a size standard to confirm complete digestion.

== Purification of Linearized DNA ==

To recover the linearized plasmids with Qiagen MinElute PCR Purification Kit:

#Add 475 uL of Buffer PBI to 95 uL of remaining linearization reaction mix.
#Place in a MinElute column in a 2 mL collection tube.
#To bind DNA, apply the sample to the QIAquick column and centrifuge for 30 seconds at 13,200rpm.
#Wash MinElute column by adding 750 uL of Buffer PE and centrifuge for 30 seconds at 13,200rpm.
#Discard the flow-through and place the MinElute column back into the same tube, which should be empty. Centrifuge for an additional minute at 13,200rpm to remove any last traces of wash buffer.
#Place MinElute column in a clean 1.5 mL micro centrifuge tube
#To elute DNA, add 38 uL of Water to MinElute membrane, let column stand for 1 minute and centrifuge the column for 30 seconds at 13,200rpm.
#Quantify 1.5 uL DNA using Nanodrop©!.

== mRNA Making ==

To sythesize mRNA from linearized plasmids using Ambion messenger SP6 kit:

#Assemble the reaction at room temperature:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | Reagents
| bgcolor="#eaeaea" | Volume (uL)
|-
| bgcolor="#ffffff" | DNA (1 ug)
| bgcolor="#ffffff" | 4 (For 0.25 ug/uL DNA)
|-
| bgcolor="#eaeaea" | H20
| bgcolor="#eaeaea" | 2
|-
| bgcolor="#ffffff" | 2 x NTPs
| bgcolor="#ffffff" | 10
|-
| bgcolor="#eaeaea" | 10 x buffer
| bgcolor="#eaeaea" | 2
|-
| bgcolor="#ffffff" | Enzyme (SP6 and
RNasin)

| bgcolor="#ffffff" | 2
|-
! bgcolor="#dadada" | Total
! bgcolor="#dadada" | 20
|}

#Incubate at 37° for 2 hours in the incubator.
#Add 1 uL of DNase for 10 minutes at 37° to digest remaining DNA.

= mRNA Synthesis with Modified Nucleotides =

Modified mRNAs containing 5mCTP and Pseudo-UTP have been shown to suppress RNA-mediated innate immune activation in vivo. The HiScribe T7 ARCA mRNA Kit is capable of incorporation of 5mCTP and Pseudo-UTP, into mRNA. Up to 2.5 mM total modified nucleotides can be added into the transcription reaction without impacting the mRNA yield significantly. Other modified UTP or CTP may also be used but RNA yield will vary depending on the properties of the nucleotides. Modified GTP and ATP should not be used because they will interfere with capping and tailing efficiency. Please note modified nucleosides are not supplied with the kit. The protocol below uses 1.25 mM 5mCTP and 1.25 mM Pseudo-UTP, generating mRNA containing 50% 5mCTP and 50% Pseudo-UTP. 

#Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Assemble the reaction in the following order: 
{| border="0" width="100%" cellspacing="0" cellpadding="0"
|-
| Nuclease-free water
| to 20 µl
|-
| 2X ARCA/NTP Mix
| 10 µl
| 1 mM GTP, 4 mM ARCA, 1.25 mM CTP, 1.25 mM UTP, >1.25 mM ATP final
|-
| 5mCTP, 10 mM
| 2.5 µl
| 1.25 mM 5mCTP final
|-
| Pseudo-UTP, 10 mM
| 2.5 µl
| 1.25 mM Pseudo-UTP final
|-
| Template DNA
| X µl
| 1 μg
|-
| T7 RNA Polymerase Mix
| 2 µl
|-
| Total reaction volume
| 20 µl
|}

#Follow standard mRNA synthesis protocol.

== mRNA Purification ==

To harvest the RNA using Qiagen MinElute RNeasy Protocol For RNA cleanup:

#Adjust the sample to a volume of 100 uL with RNase-free water, add 350 uL of RLT and mix thoroughly (note: all older kits require the addition of β-ME to buffer RLT)
#Add 250 uL of ethanol (96-100 %) to the diluted RNA, and mix thoroughly by pipetting. Do not centrifuge.
#Apply sample to an RNeasy mini column placed in a 2 mL collection tube. Close the tube gently, and centrifuge for 30 s at 13,200rpm. Discard flow-through and collection tube.
#Transfer the RNeasy column into a new 2 mL collection tube. Pipet 500 uL Buffer RPE onto the RNeasy column. Close tube gently, and centrifuge 1 minute for 13,200 rpm to wash the column and discard the flow-through.
#Add 500 uL of 80% Ethanol to RNeasy column. Close tube gently and centrifuge for 1 minute at 13,200 rpm. Discard flow-through and collection tube.
#Transfer the RNeasy column into a new 2 mL collection tube. Close tube gently and centrifuge for 5 minute at 13,200 rpm to dry the RNeasy silica-gel membrane.
#To elute transfer the RNeasy column to a new 1.5 ml collection tube. Pipet 50uL of RNase-free water directly onto the RNeasy silica-gel membrane. Close the tube and centrifuge for 1 minute at 13,200 rpm.
#Immediately put collection tubes on ice, take 2 uL + 1.5ul of RNA out into two new 1.5 mL tubes.
#Quickly store the remaining RNA in -80℃.
#Run the taken-out 2 uL RNA on a gel and Quantify the 1.5 uL RNA using Nanodrop©!.
#Stored RNA can be diluted as necessary.
[[Category:Protocols]]

MRNA by In vitro Transcription - Revision history

Paul.xu at 15:44, 13 February 2022