User:Karmella Haynes/Notebook/BioBrick cloning/2015/10/08: Difference between revisions
From OpenWetWare
(Autocreate 2015/10/08 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning) |
|||
Line 6: | Line 6: | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==10/09/15== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * Cas-tone- H3 inserts into MV12 (MV11-GFP) | ||
---- | ---- | ||
''' | '''Cas-tone Project''' | ||
* STAGE 1 - pcDNA-dCas9-VP64 vector re-design | |||
** '''DONE''' - Knock-out VP64 from C-terminus - replace AscI-VP64:HA:STOP-EcoRI-XbaI with AscI-HA:STOP-XbaI dsOligo | |||
** '''DONE''' - Knock-out FLAG from N-terminus - replace SpeI-CMV:3xFLAG:NLS-SacII with PCR-amplified (SpeI)/XbaI-CMV-SpeI-NotI-SacII; use reverse primer to add SacII | |||
* STAGE 2 - Histone parts | |||
** '''DONE''' - Order primers (annotated in Benchling) | |||
** '''DONE''' PCR-amplify histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will add extra amino acids onto conserved histone N-terminal tail. Instead, use the Kozak-like seq. from the Addgene plasmid, +3 extra Kozak bases (included in primer) | |||
* STAGE 3 - Cas-fusion | |||
** '''Insert Kozak-histone parts (X/N) into dCas9 plasmid "MV12" (S/N)''' | |||
* STAGE 4 - gRNA | |||
** Put pre-existing gRNA (from luc experiment) into pSPgRNA | |||
'''Assemblies''' | |||
# H3-1-40_MV11: H3-1-40/(X/N)/129 + MV12/(S/N)/9506 | |||
# H3-1-60_MV11: H3-1-60/(X/N)/189 + " | |||
# H3-1-80_MV11: H3-1-80/(X/N)/250 + " | |||
# H3-1-116_MV11: H3-1-116/(X/N)/373 + " | |||
# H3-1-136_MV11: H3-1-136/(X/N)/433 + " | |||
{| {{table}} | |||
* Digest of vector (Fermentas FD) | |||
** SpeI/NotI | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | |||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
| 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] --> | |||
| rowspan="7" | <!-- [[Image:GelImage.jpg| | |||
|- | |- | ||
| DNA(plasmid) || | | DNA (plasmid) || 15.0 | ||
|- | |- | ||
| | | 10x buffer || 3.0 | ||
|- | |- | ||
| | | SpeI || 1.0 | ||
|- | |- | ||
| | | NotI || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 10.0 | ||
|- | |- | ||
| || | | || 30 μL --> 37°C/ ~15 min. | ||
|} | |} | ||
* Gel purification | |||
** Sigma GenElute kit. Elute & back elute w/ 25 μL elution sln. | |||
* DpnI digest & clean-up of PCR products | |||
** Add 1 μL DpnI to each PCR rxn. Incubate @ 37°C/ 5 min. | |||
** Use the Qiagen Qiaquick PCR Purification kit (cat. no. 28104 ) to purify DNA. | |||
** Elute & back-elute with 30 μL of elution solution. | |||
{| {{table}} cellspacing="3" <!-- | * Measure [DNA] for vector and inserts | ||
|- | {| {{table}} cellspacing="3" <!-- [DNA] table --> | ||
|- bgcolor=#cfcfcf | |||
| | | Sample || OD260 || 260/280 || ng/μL | ||
| | |||
|- | |- | ||
| | | 1. MV11 (S/N) || 0.03 || 1.879 || 13.5 | ||
|- | |- | ||
| | | 2. H3-1-40 PCR || 0.03 || 1.879 || 29.8 | ||
|- | |- | ||
| | | 3. H3-1-60 PCR || 0.04 || 1.974 || 39.7 | ||
|- | |- | ||
| | | 4. H3-1-80 PCR || 0.047 || 1.865 || 46.8 | ||
|- | |- | ||
| | | 5. H3-1-116 PCR || 0.06 || 1.887 || 59.57 | ||
|- | |- | ||
| | | 6. H3-1-136 PCR || 0.062 || 1.932 || 62.0 | ||
|} | |} | ||
* | * Digest & dephos inserts | ||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | |||
|- valign="top" | |||
{| {{table}} cellspacing="3" <!-- | |||
|- | |||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| DNA ( | | DNA (500 ng) || up to 15.0 μL | ||
|- | |||
| 10X FD buffer || 2.0 | |||
|- | |||
| FD EcoRI || 1.0 | |||
|- | |- | ||
| | | FD XbaI || 1.0 | ||
|- | |- | ||
| | | Roche SAP || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || x μL | ||
|- | |- | ||
| || 20 | | || 20.0 | ||
|} | |} | ||
Thermal cycler: LabNet OptiMax "AnOlig shrt" | |||
* 37°C, 10 min | |||
* 95°C, 5 min | |||
* ''Ramp down to 25°C, 5°C/1 min.'' [90/1 min, 85/1 min, 80/1 min, ... 25°C/1 min] | |||
* 25°C, ∞ | |||
* Final concentration = 25 ng/μL. | |||
* Ligations | * Ligations | ||
** > 2:1 ratio calculations... | |||
** 433 bp largest insert / 9506 bp vector * 2 * 50 ng vector = 4.5 ng | |||
** 129 bp smallest insert / 9506 bp vector * 2 * 50 ng vector = 1.4 ng | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | {| {{table}} cellspacing="3" <!-- Ligation rxn table --> | ||
|- valign="top" | |- bgcolor="#cfcfcf" valign="top" | ||
| Reagent | |||
| Rxn1-5 | |||
| | | Rxn6 | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 0.5 || --- | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 3.5 || 3.5 | ||
|- | |- | ||
| 2x lgn buf (Roche) || | | 2x lgn buf (Roche) || 5.0 || 5.0 | ||
|- | |- | ||
| T4 ligase (NEB) || 1.0 || 1.0 | | T4 ligase (NEB) || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || --- || 0.5 | ||
|- | |- | ||
| || | | || 10.0 μL || 10.0 μL | ||
|} | |} | ||
RESULTS (5/22/15) | |||
* Success! Ligation plates have 15-20 colonies, neg. ctrl has 1 colony | |||
* Pick 2 colonies from each plate for 5 mL cultures and streaks | |||
Revision as of 16:16, 8 October 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> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
10/09/15
Cas-tone Project
Thermal cycler: LabNet OptiMax "AnOlig shrt"
RESULTS (5/22/15)
|