Amlification/Cloning of Cbh Promoter
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Florian Schmidt
Sep 19, 2015, 7:57:52 PM9/19/15
to Genome Engineering using CRISPR/Cas Systems
Hello all!
thank you very much for providing support to the CRISPR-Community by this forum.
I am wondering, how the Cbh Promoter (reference: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177952/) was cloned into the SpCas9 Plasmid Addgene #48137 .
Though I tried various Primers and conditions (3-10% DMSO, 1M Betaine, GC buffer, Temperature Gradient all with Thermo Scientific Phusion HS II Polymerase) I did not manage to amplify the full length 800 bp Promoter yet.
This is most likely due to the extremely GC rich stretches upstream of the MVM Intron. At least this is the position where also sequencing reads of the Promoter (starting from SpCas9) collaps.
Furthermore, a stretch of 12 bp starting from Base No. 14 of the Promoter is repeated ~200bp downstream which might also contribute to the PCR issues.
Was the Cbh Promoter in #48137 constructed de novo from its different parts as described in Gray et al. or transfered from the original vector from Gray via cut and paste?
Or is there indeed a PCR-protocol that allows for amplification of the entire Promoter?
Thanks a lot in advance!
thank you very much for providing support to the CRISPR-Community by this forum.
I am wondering, how the Cbh Promoter (reference: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177952/) was cloned into the SpCas9 Plasmid Addgene #48137 .
Though I tried various Primers and conditions (3-10% DMSO, 1M Betaine, GC buffer, Temperature Gradient all with Thermo Scientific Phusion HS II Polymerase) I did not manage to amplify the full length 800 bp Promoter yet.
This is most likely due to the extremely GC rich stretches upstream of the MVM Intron. At least this is the position where also sequencing reads of the Promoter (starting from SpCas9) collaps.
Furthermore, a stretch of 12 bp starting from Base No. 14 of the Promoter is repeated ~200bp downstream which might also contribute to the PCR issues.
Was the Cbh Promoter in #48137 constructed de novo from its different parts as described in Gray et al. or transfered from the original vector from Gray via cut and paste?
Or is there indeed a PCR-protocol that allows for amplification of the entire Promoter?
Thanks a lot in advance!
Alex Z
Sep 20, 2015, 7:04:41 AM9/20/15
to Genome Engineering using CRISPR/Cas Systems
I am in the same situation, except I am trying to amply a GC-rich (80%) exon region.
Kai Schönig
Sep 21, 2015, 4:00:30 AM9/21/15
to Genome Engineering using CRISPR/Cas Systems
Hello,
Try to amplify the Cbh Promoter with the follwoing polymerase:KOD Xtreme™ Hot Start DNA Polymerase
First try your normal PCR protocol. If this does not work try it with the folowing step down protocol:
The ‘step down’ temperature cycling protocol with KOD polymerase:
1. 2 min at 95°C,
2. 1 s at 98°C,
3. 1 min/kb at 74°C,
4. 1 s at 98°C,
5. 1 min/kb at 72°C,
6. 1 s at 98°C,
7. 1 min/kb at 70°C,
8. 1 s at 98°C,
9. 1 min/kb at 68°C and
hold at 4°C.
repeat 5 times steps (2) and (3)
repeat 5 times steps (4) and (5)
repeat 5 times steps (6) and (7) and
repeat 5 times steps (8) and (9)
This has been working in our lab.
regards-
Kai
Yu Wang
May 25, 2016, 9:33:23 PM5/25/16
to Genome Engineering using CRISPR/Cas Systems
Same here, have you worked out the PCR?
On Saturday, September 19, 2015 at 7:57:52 PM UTC-4, Florian Schmidt wrote:
Hung Nguyen
Jun 2, 2016, 1:51:31 AM6/2/16
to Genome Engineering using CRISPR/Cas Systems
I'm also having this issue. Let us know if you've figured out the solution.
On Saturday, September 19, 2015 at 7:57:52 PM UTC-4, Florian Schmidt wrote:
Kai Schönig
Jun 2, 2016, 2:31:09 PM6/2/16
to Genome Engineering using CRISPR/Cas Systems
Did you try the PCR with the KOD Taq and the step down protocol above? This works for us to amplify part of the CAG promoter.
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