User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/21: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(fix raw html notebook nav)
 
(13 intermediate revisions by one other user not shown)
Line 2: Line 2:
|-
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
|-
|-
| colspan="2"|
| colspan="2"|
Line 9: Line 9:
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Cas-tone - Assemblies, H3's + MV11
* Cas-tone - Assemblies, H3's + MV11
* Ryan - pick pBra-BraR-MRV colonies




Line 27: Line 28:


'''Assemblies'''
'''Assemblies'''
# H3-1-40_MV11: H3-1-40/(X/N)/size + MV11/(S/N)/9506
# H3-1-40_MV11: H3-1-40/(X/N)/129 + MV11/(S/N)/9506
# H3-1-60_MV11: 5' part/(a/b)/size + MV11/(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 + "




Line 40: Line 44:
| 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|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
|-
|-
| DNA (plasmid) || up to 25 μL
| DNA (plasmid) || 15.0
|-
|-
| 10x buffer || 3.0
| 10x buffer || 3.0
|-
|-
| enzyme 1 || 1.0
| SpeI || 1.0
|-
|-
| enzyme 2 || 1.0
| NotI || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || 10.0
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 30 μL --> 37°C/ ~15 min.
|}
|}


* Gel purification
** Sigma GenElute kit. Elute & back elute w/ 25 μL elution sln.




* Digest of PCR fragments (PCR, Clip, & Clone)
** DpnI (to get rid of methylated vector)/XbaI/NotI


* 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.




 
* Measure [DNA] for vector and inserts
* Measure conc.'s
{| {{table}} cellspacing="3" <!-- [DNA] table -->
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf  
|- bgcolor=#cfcfcf  
| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 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
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| 5. H3-1-116 PCR || 0.06 || 1.887 || 59.57
|-
| 6. H3-1-136 PCR || 0.062 || 1.932 || 62.0
|}
|}




* Dephosphorylation (Roche)
* Digest & dephos inserts
{| {{table}} cellspacing="3" <!-- Dephos table -->
 
|-
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| DNA (500 ng) || up to 15.0 μL
|-
|-
| 10x buffer d.p. || 2.0
| 10X FD buffer || 2.0
|-
|-
| phosphatase || 1.0
| FD EcoRI || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| FD XbaI || 1.0
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| Roche SAP || 1.0
|-
| dH<sub>2</sub>O || x μL
|-
| &nbsp; || 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
{| {{table}} cellspacing="3" <!-- Ligations table -->
** > 2:1 ratio calculations...
|- bgcolor=#cfcfcf
** 433 bp largest insert / 9506 bp vector * 2 * 50 ng vector = 4.5 ng
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
** 129 bp smallest insert / 9506 bp vector * 2 * 50 ng vector = 1.4 ng
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
|-
| 2. vector(c/d)/ ## ng || &nbsp;
|}


{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
|- 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
|-
|-
| &nbsp;            || # μL || # μL ||
| &nbsp;            || 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


----
----
'''Oligo annealing'''
'''Ryan - pick pBra-BraR-MRV colonies"
# New BB 1
* pBra-BraR-MRV is the only receiver with no potential successes so far
# New BB 2
* Pick 8 colonies form the traditional ligation plate for  5 mL cultures (o/n) and streaks


{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
|-
| 10x annealing buffer || 2.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|}




Latest revision as of 00:58, 27 September 2017

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

05/21/15

  • Cas-tone - Assemblies, H3's + MV11
  • Ryan - pick pBra-BraR-MRV colonies


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 "MV11" (S/N)
  • STAGE 4 - gRNA
    • Put pre-existing gRNA (from luc experiment) into pSPgRNA


Assemblies

  1. H3-1-40_MV11: H3-1-40/(X/N)/129 + MV11/(S/N)/9506
  2. H3-1-60_MV11: H3-1-60/(X/N)/189 + "
  3. H3-1-80_MV11: H3-1-80/(X/N)/250 + "
  4. H3-1-116_MV11: H3-1-116/(X/N)/373 + "
  5. H3-1-136_MV11: H3-1-136/(X/N)/433 + "


  • Digest of vector (Fermentas FD)
    • SpeI/NotI
Reagent Volume
DNA (plasmid) 15.0
10x buffer 3.0
SpeI 1.0
NotI 1.0
dH2O 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.


  • Measure [DNA] for vector and inserts
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
Reagent Volume
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
dH2O x μL
  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
    • > 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
Reagent Rxn1-5 Rxn6
Insert DNA 0.5 ---
Vector DNA 3.5 3.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 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


Ryan - pick pBra-BraR-MRV colonies"

  • pBra-BraR-MRV is the only receiver with no potential successes so far
  • Pick 8 colonies form the traditional ligation plate for 5 mL cultures (o/n) and streaks



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/05/21&oldid=1016865"