*Buffer S was added to enhance the DNA’s adherence to the spin column’s wall
**Buffer SP1 was added because RNase A triggers RNA degradation
***Resuspending help increase the surface area of contact between the pellets and the reagents
****Buffer SP2 help make the mixture homogenous without breaking target DNA strands (plasmids). As
linear
bacterial chromosomes (compared to circular plasmids) are more easily dissociated and adsorbed by
proteins, they will also form precipitates but circular plasmids will not.
*****Buffer SP3 is a buffer with strong acidity that will separate the testing solution into two
distinct
sections: a turbid mix of complex proteins on the top and a clear solution of DNA on the bottom.
Furthermore, its acidity neutralizes the solution and prevents alkali from causing DNA degradation.
Same procedure as “B. Test OD value of pET-28a integrated E. coli (dedicated to produce Cas12a protein)”, except set ddH2O as the controlled group.
Results of the test are shown in the table below:
Integrated Bacterial Sequence | Data collected | |||
---|---|---|---|---|
ng/μl * | A260/A280** | A260/A230*** | A260**** | |
invA | 270.5 | 1.93 | 2.16 | 5.411 |
16S | 205.4 | 1.98 | 2.12 | 4.109 |
cagA | 430.2 | 1.87 | 2.23 | 8.604 |
ipaH | 333.7 | 1.94 | 2.13 | 6.674 |
* ng/μl: DNA concentration
** A260/A280: bacteria concentration
*** A260/A230: DNA purity, a value between 1.8 and 2.0 are considered as normal
**** A260: RNA residue, a value approximate to 2 is normal
Number of Primer(s) | ||||||
---|---|---|---|---|---|---|
Integrated sequence |
sgDNA(1)* forward |
sgDNA(1) reverse |
sgDNA(2) forward |
sgDNA(2) reverse |
oligoDNA** forward |
oligoDNA reverse |
16S | T7-F | 16S(-)1 | T7-F | 16S(-)2 | 16S-F | 16S-R |
cagA | T7-F | cagA(-)1 | T7-F | cagA(-)2 | cagA-F | cagA-R |
invA | T7-F | invA(-)1 | T7-F | invA(-)2 | invA-F | invA-R |
ipaH | T7-F | ipaH(-)1 | T7-F | ipaH(-)2 | ipaH-F | ipaH-R |
*Two sets of sgDNAs will be used for each plasmid sequence. However, both will use the same forward
primer. This is to compare and contrast the efficiency of primers of different lengths.
**oligo DNA: forward and reverse primers start at the 5’ end of the upper and the lower strand of
the integrated sequence. This set is to prove that the viral DNA has been integrated successfully
into the plasmid.
Total Volume | 120 μl |
2× concentrated MasterMix | 60μl |
ddH2O | 42μl |
Primer-forward | 6μl |
Primer-reverse | 6μl |
Template(plasmid) | 6μl |
(2 possible candidates for sgRNA in the form of DNA and 1 oligoDNA for each of the four strands will undergo amplification)
*agarose: agarose creates a sieve that separates DNA with nuance length differences
**TAE: TAE buffer provides ions during the electrophoresis. We prepared the 300ml of the solution by
adding the following reagents together in the following ratio: TAE: ddH2O = 1:50
*sgRNA 1a and 1b are obtained from the same tube. The same is for 2a and 2b.
Agar Mass (g) and volume of Buffer B2 (μl) added
Oligo DNA | SgRNA 1 | SgRNA 2 | |
---|---|---|---|
cagA | 0.30g: 900 μl | 0.670g: 250 μl (Actual) | 0.408g: 1200 μl |
16S | 0.4078: 1200 μl | 0.311g: 900 μl | 0.767g: 2000 μl |
invA | 0.306g: 900 μl | 0.327g: 1000 μl | 0.623g: 1850 μl |
ipaH | 0.201g: 1000 μl | 0.423g: 1360 μl | 0.542g: 1600 μl |
Same procedure as “B. Test OD value of pET-28a integrated E. coli (dedicated to producing Cas12a protein)”, except set Elution Buffer as the control group before testing the concentration
The test results are shown in the table below:
cagA | 16S | invA | ipaH | |
---|---|---|---|---|
Oligo DNAs | 38.65 | 108.1 | 64.95 | 4.9 |
SgRNA 1 | 12.75 | 0.4 | 20.75 | 28.4 |
SgRNA 2 | 11.3 | 17.7 | 17.84 | 15.8 |
Name of Reagent/Template | SgRNA1 cagA | SgRNA2 cagA | SgRNA1 ipaH | SgRNA2 ipaH | SgRNA1 invA | SgRNA2 invA | SgRNA1 16S | SgRNA2 16S |
---|---|---|---|---|---|---|---|---|
T7 Transcription Kit added(μl) | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
T7 RNA Polymerase added(μl) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
RNase Free Water added(μl) | 4 | 4 | 3 | 3 | 3 | 3 | 9 | 3 |
Template added(μl) | 5 | 5 | 6 | 6 | 6 | 6 | 0 | 6 |
Results are shown in the graph below:
Test result/Template | SgRNA1 cagA | SgRNA2 cagA | SgRNA1 ipaH | SgRNA2 ipaH | SgRNA1 invA | SgRNA2 invA | SgRNA1 16S | SgRNA2 16S |
---|---|---|---|---|---|---|---|---|
Concentration (ng/ μl) | 3197.0 | 2871.3 | 2702.2 | 3116.0 | 3169.0 | 2368.7 | 3696.1 | 2759.1 |
A260/A280 | 1.97 | 1.96 | 1.95 | 1.99 | 1.98 | 1.97 | 2.00 | 1.93 |
A260/A230 | 2.34 | 2.33 | 2.27 | 2.39 | 2.37 | 2.27 | 2.37 | 2.16 |
* DNA in vitro transcription reagent group:
Same procedure as “B. Test OD value of pET-28a integrated E. coli (dedicated to produce Cas12a protein)”, except set ddH2O as the controlled group.
Test results:
ng/ μL | A260/A280* | |
---|---|---|
16S sgRNA 1 | 3.1 | 1.20 |
16S sgRNA 2 | 11.5 | 1.04 |
invA sgRNA 1 | 10.3 | 1.62 |
invA sgRNA 2 | 14.7 | 1.62 |
ipaH sgRNA 1 | 12.5 | 2.11 |
ipaH sgRNA 2 | 13.6 | 1.53 |
cagA sgRNA 1 | 461.5 | 1.04 |
cagA sgRNA 2 | 12.4 | 1.64 |
*A260/A280: a ratio near 1.60 depict a good result