User:Karmella Haynes/Notebook/BioBrick cloning/2015/01/22: Difference between revisions
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* | * Planning: Gal4 fusion cloning | ||
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'''Planning: Gal4 fusion cloning'''<br> | |||
* "Backbone" = Gal4-mCherry-VP64 construct KAH60/pcVN | |||
* Approach: swap-in activator domain to replace VP64 | |||
** PCR-amplify backbone (omitting VP64) | |||
** PCR-amplify insert | |||
** Plan A: use LCR to ligate insert into backbone | |||
'''Primers''' (ordered 01/02/15)<br> | |||
Attempt 1: swap-in ATF activation domain; add 5' phosphates to all oligos EXCEPT for bridging oligos | |||
* KAH60 "backbone" (without VP64, includes scars) = 6637 | |||
** KAH60 F1 | |||
** KAH60 R1 | |||
* ATF2 insert = 906 | |||
** ATF2_f1 | |||
** ATF2_r1 | |||
* LCR Bridging oligos | |||
** LCRb_KAH60_ATF2_1 | |||
** LCRb_KAH60_ATF2_2 | |||
'''PCR-verify fragment primers'''<br> | |||
Try different cDNA libraries for the ATF2 fragment to account for any mutations or deletions | |||
# KAH60 "backbone" (without VP64, includes scars) = 6637: KAH60 F1/ KAH60 R1 | |||
# U2OS ATF2 insert = 906: ATF2_f1/ ATF2_r1 | |||
# SKNSH " | |||
# K562 " | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | |||
|- valign="top" | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Rxn1 | |||
| bgcolor=#cfcfcf | Rxn2 | |||
| rowspan="7" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] --> | |||
|- | |||
| Template || 1.0 KAH60/pcVN || 1.0 1:1000 U2OS cDNA || 1.0 1:1000 SKNSH cDNA || 1.0 1:1000 K562 cDNA | |||
|- | |||
| 10 uM fwd primer || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| 10 uM rev primer || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| 2x GoTaq green || 12.5 || 12.5 || 12.5 || 12.5 | |||
|- | |||
| dH<sub>2</sub>O || --- | |||
|- | |||
| || 25 μL --> 37°C/ ~30 min. | |||
|} | |||
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Revision as of 11:55, 22 January 2015
Karmella's BioBrick Cloning | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||
mm/dd/yy
Planning: Gal4 fusion cloning
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Reagent | Volume | Expected: 1. BB 1 = size 2. BB2 = size |
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DNA(plasmid) | 2.0 μL | ||||||||||||||||||||||||||||
10X buffer | 1.5 | ||||||||||||||||||||||||||||
EcoRI | 1.0 | ||||||||||||||||||||||||||||
PstI | 1.0 | ||||||||||||||||||||||||||||
dH2O | 9.5 | ||||||||||||||||||||||||||||
15 μL --> 37°C/ ~15 min. |
Assemblies
- BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
- BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
- Digests (Fermentas FD)
- Specific notes
Reagent | Volume | |
DNA (plasmid) | up to 25 μL | |
10x buffer | 3.0 | |
enzyme 1 | 1.0 | |
enzyme 2 | 1.0 | |
dH2O | --- | |
30 μL --> 37°C/ ~30 min. |
- Measure conc.'s
Sample | OD260 | 260/280 | ng/μL |
1. Digested part (a/b) | --- | --- | --- |
2. Digested part (c/d) | --- | --- | --- |
- Dephosphorylation (Roche)
Reagent | Volume |
DNA (clean digest) | up to 17 μL (500 ng) |
10x buffer d.p. | 2.0 |
phosphatase | 1.0 |
dH2O | --- |
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL |
- Ligations
Ligation | Plate results (lig : neg crtl) mm/dd/yy |
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng | new BioBrick #:1 (Pick #) |
2. vector(c/d)/ ## ng |
1 | 2 | ||
Insert DNA | ### | --- | |
Vector DNA | ### | ### | |
2x lgn buf (Roche) | ### | ### | |
T4 ligase (NEB) | 1.0 | 1.0 | |
dH2O | ### | ### | |
# μL | # μL |
Oligo annealing
- New BB 1
- New BB 2
DNA (oligos, 100 μM) | up to 18 μL (3 μL ea.) |
10x annealing buffer | 2.0 |
dH2O | --- |
20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight |
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