Differences
This shows you the differences between two versions of the page.
| Both sides previous revision Previous revision | |||
|
interactome:experiments:20191210-3 [2019/12/11 12:46] dan [Table] |
interactome:experiments:20191210-3 [2019/12/11 12:46] (current) dan |
||
|---|---|---|---|
| Line 1: | Line 1: | ||
| ## Personal Interactome | ## Personal Interactome | ||
| - | ## One-by-one pENTRY clone generation from HEK293Ta cDNA sample | + | # One-by-one pENTRY clone generation from HEK293Ta cDNA sample |
| == BP cloning== | == BP cloning== | ||
| - | #### Material | + | === Material === |
| * pExpression plasmid sample (eg. pAD-ORF) or PCR product with attB sequencing flanking the desired gene fragment. | * pExpression plasmid sample (eg. pAD-ORF) or PCR product with attB sequencing flanking the desired gene fragment. | ||
| * pDONR plasmid sample (pDONR221, pDONR223, etc.) | * pDONR plasmid sample (pDONR221, pDONR223, etc.) | ||
| Line 28: | Line 28: | ||
| - | #### Protocol | + | === Protocol=== |
| - | ====1 Substrate preparation==== | + | ==1 Substrate preparation== |
| * Prepare 6-60ng/ul solution of attB PCR sample/ plasmids. | * Prepare 6-60ng/ul solution of attB PCR sample/ plasmids. | ||
| Line 36: | Line 36: | ||
| * Add 2ul of the DNA as follows into one PCR plate. | * Add 2ul of the DNA as follows into one PCR plate. | ||
| - | ====2 BP reaction==== | + | ==2 BP reaction== |
| *Prepare Ice with a tube labled "BP" | *Prepare Ice with a tube labled "BP" | ||
| Line 56: | Line 56: | ||
| - | ====3 Transformation==== | + | ==3 Transformation== |
| *Prepare the reaction mix and competent cells on ice. | *Prepare the reaction mix and competent cells on ice. | ||
| *For each sample, add 10µl of homebrew competent NEB5-α cells. | *For each sample, add 10µl of homebrew competent NEB5-α cells. | ||