Editing FLP Recombinase mRNA (section)

=== RNA Synthesis Protocols ===

1. Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to bottom of tubes. Keep on ice.

2. If you are planning to run many reactions, it is convenient to prepare a master mix by combining equal volumes of the 10X reaction buffer and four ribonucleotide (NTP) solutions. Use 10 µl per reaction.

3. Assemble the reaction at room temperature in the following order:

'''This protocol is for DNA template with T7 promoter + IRES + GOI + polyA, such as Flp PCR product'''

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Components
| Vol
| Final Concentration/Amount
|-
| Nuclease-free water&nbsp;&nbsp;<br/>
| X µl<br/>
| <br/>
|-
| 10X Reaction Buffer<br/>
| 2 µl&nbsp;<br/>
| <br/>
|-
| ATP (100 mM)&nbsp;<br/>
| 2 µl&nbsp;<br/>
| 10 mM<br/>
|-
| GTP (100 mM)<br/>
| 2 µl&nbsp;<br/>
| 10 mM<br/>
|-
| UTP (100 mM)&nbsp;<br/>
| 2 µl&nbsp;<br/>
| 10 mM<br/>
|-
| CTP (100 mM)&nbsp;<br/>
| 2 µl&nbsp;<br/>
| 10 mM<br/>
|-
| 10 mM final Template DNA&nbsp;<br/>
| X µl&nbsp;&nbsp;<br/>
| 1 µg<br/>
|-
| T7 RNA Polymerase Mix<br/>
| 2 µl<br/>
| <br/>
|-
| Total reaction volume<br/>
| 20 µl&nbsp;<br/>
| <br/>
|}

4. Mix thoroughly, pulse-spin in microfuge. Incubate at '''37°C for 2 hours'''. The yield will not be compromised if the incubation temperature is within the range of 35–40°C.&nbsp;&nbsp;For reactions with transcripts longer than 0.3&nbsp;kb, 2&nbsp;hour incubation should give you the maximum yield.

5. Optional: DNase treatment to remove DNA template. Standard reactions normally generate large amounts of RNA at concentrations up&nbsp;to 10 mg/ ml. As a result the reaction mixture is quite viscous. It is easier to perform DNase treatment after the reaction mixture is diluted. To remove template DNA, add 70 μl nuclease-free water, 10 μl of 10X DNase I Buffer, and 2 μl of DNase I (RNase-free), mix and incubate for 15&nbsp;minutes at 37°C.

6.&nbsp;Spin columns can be used to remove unincorporated nucleotides, proteins and salts. Adjust the volume of the reaction mixture to 100 μl by adding 80 μl nuclease-free water, mix well. Purify the RNA by following the manufacturer’s instructions. Each reaction could produce up to 180 μg of RNA which may exceed column capacity thus requiring additional columns.&nbsp;

7.&nbsp;A NanoDrop Spectrophotometer can read RNA concentrations from 10 ng/μl to 3000 ng/μl directly. For single-stranded RNA, 1 A260 is equivalent to RNA concentration of 40 μg/ml. The RNA concentration can be calculated as follows: A260 x dilution factor x 40 = __ μg/ml RNA