SEA-PHAGES | All posts created by libby1954

Link to this post \| posted 12 Apr, 2018 22:14
libby1954	We are having difficulty with annotation of Bones_4. This gene is in Pham 40723, and there are only 12 members of this pham, 10 of them are annotated as a hypothetical protein. Aligning the protein sequences that are from the Phamerator maps from the 12 phages in ClustalW gives the attached alignment. Bones has a long insert, and 4 of the sequences are very different from the other 8 sequences. GeneMark shows low coding potential. Protein BLAST in NCBI shows only matches with the same 12 phages. Starterator shows 8 tracks for the 12 phages in the Pham, and Bones_4 is in its own track with a unique start site. If this is not a gene, then there is a gap of 411 bp. However, all 10 of the annotated genes call it as a gene. Our conclusion is that this is a gene, but we would like any input from the experienced annotators. 301Kb

Link to this post | posted 12 Apr, 2018 22:14

libby1954

We are having difficulty with annotation of Bones_4. This gene is in Pham 40723, and there are only 12 members of this pham, 10 of them are annotated as a hypothetical protein. Aligning the protein sequences that are from the Phamerator maps from the 12 phages in ClustalW gives the attached alignment. Bones has a long insert, and 4 of the sequences are very different from the other 8 sequences.
GeneMark shows low coding potential. Protein BLAST in NCBI shows only matches with the same 12 phages. Starterator shows 8 tracks for the 12 phages in the Pham, and Bones_4 is in its own track with a unique start site.
If this is not a gene, then there is a gap of 411 bp. However, all 10 of the annotated genes call it as a gene. Our conclusion is that this is a gene, but we would like any input from the experienced annotators.

Posted in: Gene or not a Gene → Cluster A1 gene question

Link to this post \| posted 06 Dec, 2017 19:12
libby1954	All of our 8 students at Salish Kootenai College had degraded DNA when the uncut and enzyme digests were run on a gel. It appeared to be completely degraded, since there was only a small spot near the bottom of the gel. Only one student had some faint bands at the high weight at the size of uncut. I suspect that the DNAse was not inactivated, or that too much was added. One problem was when mixing up the DNAse (I did not use RNAse). The recipe card on page 86 of Phage Discovery Instructor's Guide, 2017 Edition, was unclear. In step 4 of "To Prepare" the final volume is brought to 5 mL. But the addition of the volumes of reagents listed (including water) equals about 9.3 mL. I have not seen any posts this year about problems with degraded DNA, but wonder if there are any recommendations. We have not tried the DNA isolation again yet, and the quarter is ending this week, so we will try again in January. Another note, in the Wizard kit, we received two packages of syringe plungers, but no syringe barrels. Luckily we had other syringes we could use. Thank you, Libby

Link to this post | posted 06 Dec, 2017 19:12

libby1954

All of our 8 students at Salish Kootenai College had degraded DNA when the uncut and enzyme digests were run on a gel. It appeared to be completely degraded, since there was only a small spot near the bottom of the gel. Only one student had some faint bands at the high weight at the size of uncut.

I suspect that the DNAse was not inactivated, or that too much was added. One problem was when mixing up the DNAse (I did not use RNAse). The recipe card on page 86 of Phage Discovery Instructor's Guide, 2017 Edition, was unclear. In step 4 of "To Prepare" the final volume is brought to 5 mL. But the addition of the volumes of reagents listed (including water) equals about 9.3 mL.

I have not seen any posts this year about problems with degraded DNA, but wonder if there are any recommendations. We have not tried the DNA isolation again yet, and the quarter is ending this week, so we will try again in January.

Another note, in the Wizard kit, we received two packages of syringe plungers, but no syringe barrels. Luckily we had other syringes we could use.

Thank you,
Libby

Posted in: Phage Discovery/Isolation → Yield and degradation of DNA isolated by new protocol

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