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In vitro transcription
Parts
We constructed a standardized plasmid containing sgRNA and provided the corresponding experimental manual to provide references for other teams with similar needs.
In vitro transcription
In vitro transcription of the plasmid containing template sgDNA
1.Reagents
• Template (10ng/μL)
• Primers 1 (10μM)
• Primers 2(10μM)
• 2×Mix
• Sterile water
• Primers 1 (10μM)
• Primers 2(10μM)
• 2×Mix
• Sterile water
2. Experiments operation
1)Dilute and dissolve the template
2)Construct PCR reaction system:1μL template，25μL mix, 20μL Sterile water, 2μL primers 1，2μLprimers 2
3)Mix and put it in the PCR instrument, set procedures, and start the reaction
4)Electrophoresis Gel recovery
2)Construct PCR reaction system:1μL template，25μL mix, 20μL Sterile water, 2μL primers 1，2μLprimers 2
3)Mix and put it in the PCR instrument, set procedures, and start the reaction
4)Electrophoresis Gel recovery
In vitro transcription of sgRNA
1.Reagents
• Nucleic acid-free water
• 10×Reaction buffer
• 100mM ATP
• 100mM GTP
• 100mM UTP
• 100mM CTP
• DNA template
• T7RNA polymerase mixture
• 10×Reaction buffer
• 100mM ATP
• 100mM GTP
• 100mM UTP
• 100mM CTP
• DNA template
• T7RNA polymerase mixture
2. Experiments operation
1)Construct the transcription system
2)Incubate the samples under 37 ℃ in the PCR instrument for 4 h
3)Take 6 μL of a post-transcriptional system for electrophoresis. Use marker(2000bp) of 3μL.
2)Incubate the samples under 37 ℃ in the PCR instrument for 4 h
3)Take 6 μL of a post-transcriptional system for electrophoresis. Use marker(2000bp) of 3μL.
Purify of sgRNA
1.Reagents
• RK solution (BufferRK)
• Washing liquid RW RW(Buffer RW)
• RNase-Free ddHz0
• RNase-Free Columns CR2 set
• RNase-Free Centrifuge tubes (1.5 ml))
• β-ME
• Ethanol
• sgRNA after PCR reaction
• Washing liquid RW RW(Buffer RW)
• RNase-Free ddHz0
• RNase-Free Columns CR2 set
• RNase-Free Centrifuge tubes (1.5 ml))
• β-ME
• Ethanol
• sgRNA after PCR reaction
2. Experiments operation
1)If it is the first use, add ethanol into washing liquid RW.
2)Adding β-ME into RK to the final concentration of 1%
3)Take 14μL of the system after transcription and add 86μL RNase-Free water to 100μL. Then add 350μL RK after mixing and mix well
4)Add 250 μL ethanol, fully mix and immediately carry on to the next step
5)Add 500 μL RW to adsorption column CR2. Set under room temperature and then centrifuged at 12,000 rpm for 30s. Discard waste liquid and put CR2 into the collection tube
6)Repeat 4)
7)Centrifuge the sample under 12,000 rpm for 5 min and remove residual liquid
8)Put column CR2 into a new centrifuge tube, add 20μL RNase-Free water, and place it under room temperature for 2 min. Then centrifuge under 12000 rpm for 2 min and obtain the target solution.
9)Detect the nucleic acid concentration
2)Adding β-ME into RK to the final concentration of 1%
3)Take 14μL of the system after transcription and add 86μL RNase-Free water to 100μL. Then add 350μL RK after mixing and mix well
4)Add 250 μL ethanol, fully mix and immediately carry on to the next step
5)Add 500 μL RW to adsorption column CR2. Set under room temperature and then centrifuged at 12,000 rpm for 30s. Discard waste liquid and put CR2 into the collection tube
6)Repeat 4)
7)Centrifuge the sample under 12,000 rpm for 5 min and remove residual liquid
8)Put column CR2 into a new centrifuge tube, add 20μL RNase-Free water, and place it under room temperature for 2 min. Then centrifuge under 12000 rpm for 2 min and obtain the target solution.
9)Detect the nucleic acid concentration
3.Results
Results of purification RNA are good（A260/A280）
Standardization
Purpose:
We want to verify that the standardized plasmid can enter the experimental bacteria through the normal laboratory
transformation steps and complete the corresponding amplification steps, so as to achieve the purpose of signal amplification.
Therefore, in this experiment, our experimental design is carried out according to the standard plasmid transformation steps,
from PCR amplification, enzyme digestion, enzyme ligation to transformation into the experimental bacteria,
and subsequent verification. We focus on the conversion efficiency in each step and hope to improve the enzyme digestion
efficiency within the allowable range of experimental error as much as possible.
Results:
Column recovery
A1: PCR product
A2：enzymatically digested product
A3: uncut plasmid
A4：enzymatically digested plasmid
A5: column recovered product
A6: column recovered plasmid
Results of gel electrophoresis after column recovery are shown on the left,
yielding results that illustrate that the process of column recovery,
performed with high efficiency, has reached the experimental purpose of the column recovery step.
Enzyme-linked efficiency test
A1 : enzyme-linked product
Gel electrophoresis revealed self-ligation of a portion of the PCR fragment.
Bands with large fragments were less fluorescent,
although successful ligation of PCR products and plasmids could be demonstrated.
Results of transformed colony culture
The left panel shows the growth of colonies after overnight incubation, and it can be seen that a single colony grows on both media used experimentally.
Enzymatic cleavage result verification
A1: uncleaved plasmid ①
A2: digested product ①
B1: uncut plasmid ②
B2: digested product ②
A total of two single colonies were picked for shaker culture in the experiment,
which are designated ①, ②. Since the plasmid is circular when uncut,
electrophoresis is faster; Became linear after cutting, and electrophoresis was slower.
Therefore, ② is more consistent with our experimental expectations.
Sequencing result: the uncut plasmid ② was sent for sequencing,
and the sequencing result was much different from the expectation.
Speculated that the reason might be that the PCR products self-associate during the enzyme-linked process,
which caused the transformation. The plasmid concentration can be appropriately increased and
the PCR fragment concentration decreased when the next experiment is performed.
Sequencing results
This is the DNA sequence we designed before the experiment. Its length is 88 BP. We hope that this gene sequence can still be
extracted after a series of biological operations such as inserting the fragment into the plasmid and transferring it into the experimental bacteria
for amplification and culture.
T A A T A C G A C T C A C T A T A G G G G A A A T T A A T A C G A C T C A C T A T A G G G A A T T T C T A C T G T T G T A G A T T A G C T T A T C A G A C T G A T G T T G A T T
We sent the results of the experiment to the company for DNA sequencing. The results showed that our experiment was successful.
From the sequencing results, we can see that a small fragment with a length of about 88 bp has been produced,
which proves that we have completed a series of transformation processes such as plasmid extraction, and enzyme ligation and enzyme digestion.
