The number of base pairs for the PGEX-KG (original) plasmid and PGEX-KG SAW1 (clone gene) is different.
PGEX-KG- Saw1 has 794 bp more than original plasmid.
The single digestion for both PGEX-KG and PGEX-KG-Saw1 will make a single cut in their respective restriction sites. The clone and original plasmids will become linearized and the PGEX-KG-Saw1 will be longer than PGEX-KG because of the insertion of Saw1. However, the insertion Saw1 can be isolated from the clone plasmid by doing double digestion with restriction enzymes EcorI and HindIII. The restriction sites include the Saw1 insertion sequence when the particular enzymes are used. Such Saw1 fragment will not be found original PGEX-KG plasmid.
The orientation of saw1 insertion cannot …show more content…
There will be three assays will be made and tested with both known and unknown plasmid.
The first assay contains both PGEX-F1 primer and PGEX-R primer. Both primers enable to do replication with PCR in both Watson(top) strand and Crick(bottom). The presence of Saw1 insertion gene will not matter in this assay. Therefore, the known and unknown will not show a difference in bands generated from the PCR and Gel electrophoresis.
The second assay has PGEX-F1 primer and Saw1 primer. The Saw1 primer can either act as forward primer or reverse primer depends on the orientation of Saw1 gene insertion According to the manual, Saw1 acts as a forward primer for reverse plasmid and reverse primer for forward plasmid. Essentially the saw1 gene is helping to identify the orientation of the unknown plasmid. The saw1 primer can’t act of original known plasmid which lacks of saw1 insertion.
The third assay has both reverse primer PGEX-R1 and saw1 primer. This assay also helps to identify the orientation of unknown plasmid because of Saw1 primer’s role …show more content…
Therefore, there will be a difference in target nucleic acid presence and the CT value. The CT (threshold cycle) obtained from real-time PCR for both known and unknown (#4) were
Known- 31.37
Unknown -35.3
The higher CT value (over 29) indicated lower amount of target nucleic acid which correlated with less number amplification in qPCR. Since the values are closer they strongly suggest unknown # 4 is in reverse orientation.
8. Both restriction digest and PCR are essential extracting the plasmid information. However, restriction digest and PCR didn’t agree with each other because they give different information about plasmid. The restriction digest reveals weather the unknown plasmid has an insertion sequence(Saw1) or not. The PCR tells the insertion sequence’s orientation. Since the PCR experiment did not provide analyzable information, the experiment result is
PGEX-KG- Saw1 has 794 bp more than original plasmid.
The single digestion for both PGEX-KG and PGEX-KG-Saw1 will make a single cut in their respective restriction sites. The clone and original plasmids will become linearized and the PGEX-KG-Saw1 will be longer than PGEX-KG because of the insertion of Saw1. However, the insertion Saw1 can be isolated from the clone plasmid by doing double digestion with restriction enzymes EcorI and HindIII. The restriction sites include the Saw1 insertion sequence when the particular enzymes are used. Such Saw1 fragment will not be found original PGEX-KG plasmid.
The orientation of saw1 insertion cannot …show more content…
There will be three assays will be made and tested with both known and unknown plasmid.
The first assay contains both PGEX-F1 primer and PGEX-R primer. Both primers enable to do replication with PCR in both Watson(top) strand and Crick(bottom). The presence of Saw1 insertion gene will not matter in this assay. Therefore, the known and unknown will not show a difference in bands generated from the PCR and Gel electrophoresis.
The second assay has PGEX-F1 primer and Saw1 primer. The Saw1 primer can either act as forward primer or reverse primer depends on the orientation of Saw1 gene insertion According to the manual, Saw1 acts as a forward primer for reverse plasmid and reverse primer for forward plasmid. Essentially the saw1 gene is helping to identify the orientation of the unknown plasmid. The saw1 primer can’t act of original known plasmid which lacks of saw1 insertion.
The third assay has both reverse primer PGEX-R1 and saw1 primer. This assay also helps to identify the orientation of unknown plasmid because of Saw1 primer’s role …show more content…
Therefore, there will be a difference in target nucleic acid presence and the CT value. The CT (threshold cycle) obtained from real-time PCR for both known and unknown (#4) were
Known- 31.37
Unknown -35.3
The higher CT value (over 29) indicated lower amount of target nucleic acid which correlated with less number amplification in qPCR. Since the values are closer they strongly suggest unknown # 4 is in reverse orientation.
8. Both restriction digest and PCR are essential extracting the plasmid information. However, restriction digest and PCR didn’t agree with each other because they give different information about plasmid. The restriction digest reveals weather the unknown plasmid has an insertion sequence(Saw1) or not. The PCR tells the insertion sequence’s orientation. Since the PCR experiment did not provide analyzable information, the experiment result is