User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/17: Difference between revisions

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|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}}}}]]}}
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==mm/dd/yy==
==04/17/15==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Rene - gRNA plasmids, 5 mL cultures (11 total)
* Line item 2
* Ryan - Receiver plasmids, assembly
* LCR Development - Repeat Phusion for MV10, use correct amount of 5x buffer




----
----
'''Minipreps'''<br>
'''Ryan - Receiver plasmids, assembly'''
* Check with E/P digests
* Stage 1 - insert Regulator ORFS
** Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
** Insert ORF(E/X) into Vector (E/X). see [https://benchling.com/s/VFNzuauP/edit Vector plasmid map]
* Stage 2 - insert Promoters
** Cut & dephos promoters with E/X
** Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
Phusion PCR
# AubR, EcoRI F/ XbaI R
# BjaR, "
# BraR, "
# RpaR, "
 
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| bgcolor=#cfcfcf | Mix (x5)
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
| rowspan="7" | <u>Expected:</u><br>1. AubR = 851<br>2. BjaR = 776<br>3. BraR = 776<br>4. RpaR = 779<br>
| rowspan="7" | [[Image:KAH041715_gel1.jpg|200px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA(plasmid) || 2.0 μL
| DNA(plasmid) || 0.5 μL || ---
|-
|-
| 10X buffer || 1.5
| 10 μM F primer || 1.0 || 5.0
|-
|-
| EcoRI || 1.0
| 10 μM R primer || 1.0 || 5.0
|-
|-
| PstI || 1.0
| 10 mM dNTPs || 1.0 || 5.0
|-
|-
| dH<sub>2</sub>O || 9.5
| Phusion Pol. || 0.5 || 2.5
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| 5X HF buffer || 10.0 || 50.0
|-
| dH<sub>2</sub>O || 36.0 || 180.0
|-
| &nbsp; || 50.0 ||
|}
|}
Program: Phusion (block B)
* 98°C, 3 min
* 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec]
* 72°C, 10 min
* 4°C ∞
Conclusion
* All four worked. Proceed to next step


----
----
'''Assemblies'''
'''LCR Development'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
* Repeat Phusion for MV10 (from 4/16/15), use correct amount of 5x buffer
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size




* Digests (Fermentas FD)
Phusion PCR
** Specific notes


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Rxn1,2
| 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" | <u>Expected:</u><br>1,2. MV10 = 5191
| rowspan="7" | [[Image:KAH041715_gel2.jpg|150px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA (plasmid) || up to 25 μL
| DNA(clean linear) || 1.0 μL
|-
|-
| 10x buffer || 3.0
| 10 μM F primer || 1.0
|-
|-
| enzyme 1 || 1.0
| 10 μM R primer || 1.0  
|-
|-
| enzyme 2 || 1.0
| 10 mM dNTPs || 1.0  
|-
|-
| dH<sub>2</sub>O || ---
| Phusion Pol. || 0.5
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| 5X HF buffer || 10.0
|}
 
 
* Measure conc.'s
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| dH<sub>2</sub>O || 35.5
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| &nbsp; || 50.0 ||  
|}
|}




* Dephosphorylation (Roche)
Program: Phusion (block A)
{| {{table}} cellspacing="3" <!-- Dephos table -->
* 98°C, 3 min
|-
* 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min]
| bgcolor=#cfcfcf | Reagent
* 72°C, 10 min
| bgcolor=#cfcfcf | Volume
* 4°C ∞
|-
| DNA (clean digest) || up to 17 μL (500 ng)
|-
| 10x buffer d.p. || 2.0
|-
| phosphatase || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|}


Conclusion
* Big improvement. Keep this for potential cloning
* Also try again with higher annealing temp


* Ligations
{| {{table}} cellspacing="3" <!-- Ligations table -->
|- bgcolor=#cfcfcf
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
|-
| 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 -->
| &nbsp;            || 1    || 2    ||
|-
| Insert DNA        || ###  || ---  ||
|-
| Vector DNA        || ###  || ###  ||
|-
| 2x lgn buf (Roche) || ###  || ###  ||
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
| dH<sub>2</sub>O    || ###  || ###  ||
|-
| &nbsp;            || # μL || # μL ||
|}
----
'''Oligo annealing'''
# New BB 1
# New BB 2
{| 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 00:55, 27 September 2017

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

04/17/15

  • Rene - gRNA plasmids, 5 mL cultures (11 total)
  • Ryan - Receiver plasmids, assembly
  • LCR Development - Repeat Phusion for MV10, use correct amount of 5x buffer


Ryan - Receiver plasmids, assembly

  • Stage 1 - insert Regulator ORFS
    • Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
    • Insert ORF(E/X) into Vector (E/X). see Vector plasmid map
  • Stage 2 - insert Promoters
    • Cut & dephos promoters with E/X
    • Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


Phusion PCR

  1. AubR, EcoRI F/ XbaI R
  2. BjaR, "
  3. BraR, "
  4. RpaR, "
Reagent Volume Mix (x5) Expected:
1. AubR = 851
2. BjaR = 776
3. BraR = 776
4. RpaR = 779
 Hover name
5 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.5 μL ---
10 μM F primer 1.0 5.0
10 μM R primer 1.0 5.0
10 mM dNTPs 1.0 5.0
Phusion Pol. 0.5 2.5
5X HF buffer 10.0 50.0
dH2O 36.0 180.0
  50.0


Program: Phusion (block B)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • All four worked. Proceed to next step


LCR Development

  • Repeat Phusion for MV10 (from 4/16/15), use correct amount of 5x buffer


Phusion PCR

Reagent Rxn1,2 Expected:
1,2. MV10 = 5191 		Hover name
15 μL/lane; 1% agarose; Ladder
DNA(clean linear) 1.0 μL
10 μM F primer 1.0
10 μM R primer 1.0
10 mM dNTPs 1.0
Phusion Pol. 0.5
5X HF buffer 10.0
dH2O 35.5
  50.0


Program: Phusion (block A)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞

Conclusion

  • Big improvement. Keep this for potential cloning
  • Also try again with higher annealing temp



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