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Cicikashou (Talk | contribs) (New page: ==~~~~== So our assignment was to collect the HA, MYC, FLAG, and AP tag sequence found in vector that can be used in E.coli. I found the MYC tag. Because they are under 30bp, we decide to ...) |
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So our assignment was to collect the HA, MYC, FLAG, and AP tag sequence found in vector that can be used in E.coli. I found the MYC tag. Because they are under 30bp, we decide to do EIPCR and the whole morning me and Sherine were trying to understand EIPCR and design our oligos. We looked at the turtorial and ask Dirk and Terry about it, but I was still pretty confused because I didn't get where we should put the restricition sites and how it will work. And then Jin send us the annealing regions on the pBca1256 which causes more confusion. | So our assignment was to collect the HA, MYC, FLAG, and AP tag sequence found in vector that can be used in E.coli. I found the MYC tag. Because they are under 30bp, we decide to do EIPCR and the whole morning me and Sherine were trying to understand EIPCR and design our oligos. We looked at the turtorial and ask Dirk and Terry about it, but I was still pretty confused because I didn't get where we should put the restricition sites and how it will work. And then Jin send us the annealing regions on the pBca1256 which causes more confusion. | ||
| - | So for the forward oligo, it's insert-EcoRI-insert-Bgl-[part]-Bam-annealing region | + | So for the forward oligo, it's |
| + | <pre> | ||
| + | insert-EcoRI-insert-Bgl-[part]-Bam-annealing region | ||
| + | </pre> | ||
| + | The annealing reigon starts from the Bam site and it's about 20bp. The reverse oligo is the reverse ca1246 that Jin sent, so basically we are using the same reverse oligo evertime. I still need to do the EIPCR on Ape for more practice. But the thing is that later Jin decided that since the oligos are too long, we are going to do the wobble! So how we design the oligos is that we got the sequence: | ||
| + | <pre> | ||
| + | insert-EcoRI-insert-Bgl-[part]-Bam-insert | ||
| + | </pre> | ||
| + | and there should be 20bp overlap and GC should be b/ 40-50%. And I record my oligos to google doc and did my construction files. | ||
Revision as of 18:17, 24 June 2008
Contents |
23 June 2008
So our assignment was to collect the HA, MYC, FLAG, and AP tag sequence found in vector that can be used in E.coli. I found the MYC tag. Because they are under 30bp, we decide to do EIPCR and the whole morning me and Sherine were trying to understand EIPCR and design our oligos. We looked at the turtorial and ask Dirk and Terry about it, but I was still pretty confused because I didn't get where we should put the restricition sites and how it will work. And then Jin send us the annealing regions on the pBca1256 which causes more confusion. So for the forward oligo, it's
insert-EcoRI-insert-Bgl-[part]-Bam-annealing region
The annealing reigon starts from the Bam site and it's about 20bp. The reverse oligo is the reverse ca1246 that Jin sent, so basically we are using the same reverse oligo evertime. I still need to do the EIPCR on Ape for more practice. But the thing is that later Jin decided that since the oligos are too long, we are going to do the wobble! So how we design the oligos is that we got the sequence:
insert-EcoRI-insert-Bgl-[part]-Bam-insert
and there should be 20bp overlap and GC should be b/ 40-50%. And I record my oligos to google doc and did my construction files.
