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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==
==04/28/15==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Ryan - minipreps & digests
* Line item 2
* Ryan - PCR & Dig/Lig (promoter insert trial #2)




----
----
'''Minipreps'''<br>
'''Minipreps'''<br>
* Check with E/P digests
* Use Sigma GenElute; elute with 75 μL elution solution
* '''BraR/MRV colonies''' - picked 4 additional colonies from original cloning plate
* '''Cut w/ E/X'''
# Bra/MRV 3
# Bra/MRV 4
# Bra/MRV 5
# Bra/MRV 6
* '''promoter/Regulator/MRV colonies'''
* '''Cut w/ KpnI/EcoRI''' - BbsI drop-in of promoter destroys KpnI site; empty vector will produce 2 bands, successful clones will be linearized (1 cut)
# pAub/AubR/MRV
# pBja/BjaR/MRV
# pRpa/RpaR/MRV
 


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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| 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>
| rowspan="7" | <u>Expected:</u><br>1-4. Bra/MRV = ~3200, 776<br>Empty MRV E/X = ~3200<br>5. pAub/AubR/MRV = 4240<br>6. pBja/BjaR/MRV = 4099<br>7. pRpa/RpaR/MRV = 4116<br>Empty vector = ~4000, 971
| 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" | [[Image:KAH042815_gel1.jpg|250px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA(plasmid) || 2.0 μL
| DNA(plasmid) || 3.0 μL
|-
|-
| 10X buffer || 1.5
| 10X buffer || 1.5
|-
|-
| EcoRI || 1.0
| enzyme 1 || 1.0
|-
|-
| PstI || 1.0
| enzyme 2 || 1.0
|-
|-
| dH<sub>2</sub>O || 9.5
| dH<sub>2</sub>O || 8.5
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}
|}
* Measure [DNA]
{| {{table}}
|-
| Sample || OD 260 || 260/280 || ng/μL
|-
| BraR/MRV #5 || 0.465 || 1.913 || 465.4
|}
Conclusions
* BraR/MRV - Success! Keep colony 5 (lane 3)
* promter/Reg/MRV - none worked. Try the assembly again with PCR-amplified promoter inserts


----
----
'''Assemblies'''
'''Ryan - PCR & Dig/Lig (promoter insert trial #2)'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


'''Ryan - Receiver plasmids, assembly''' - REVISED STRATEGY (PCR promoters)
* 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
** '''PCR-amplify promoter inserts''' (same primers as Stage 1)
** '''Cut & dephos promoters with E/X'''
** '''Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling'''


* Digests (Fermentas FD)
** Specific notes


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
'''Assemblies'''
|- valign="top"
# pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
| bgcolor=#cfcfcf | Reagent
# pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
| bgcolor=#cfcfcf | Volume
# pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977
| 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] -->
# pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980
|-
| DNA (plasmid) || up to 25 μL
|-
| 10x buffer || 3.0
|-
| enzyme 1 || 1.0
|-
| enzyme 2 || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
|}




* Measure conc.'s
* Measure conc.'s
** Values from last week, except BraR/MRV, which was measured today
{| {{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. AubR/MRV || 0.258 || 1.906 || 257.7
|-
| 2. BjaR/MRV || 0.177 || 1.904 || 177.3
|-
| 3. '''BraR/MRV #5''' || 0.465 || 1.913 || 465.4
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| 4. RpaR/MRV || 0.246 || 1.896 || 246.2
|}
|}


* PCR-amplify promoters
** 2x 50 μL rxns each
# pAubR, EcoRI fwd, XbaI rev
# pBjaR, EcoRI fwd, XbaI rev
# pBraR, EcoRI fwd, XbaI rev
# pRpaR, EcoRI fwd, XbaI rev


* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- PCR rxn table -->
{| {{table}} cellspacing="3" <!-- Dephos table -->
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Vol
| bgcolor=#cfcfcf | Mix (x2)
| rowspan=7 | Expected:<br>1. pAubR = 153<br>2. pBjaR = 122<br>3. pBraR = 214<br>4. pRpaR = 136
| rowspan=7 | [[Image:KAH042815_gel2.jpg|250px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| bgcolor=#cfcfcf | Reagent
| gBlock DNA (2 ng/μL) || 0.5  || 1.0
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| 10 μM XbaI rev    || 1.0  || 2.0 
|-
|-
| 10x buffer d.p. || 2.0
| 10 μM EcoRI fwd    || 1.0  || 2.0  
|-
|-
| phosphatase || 1.0
| 2x GoTaq green    || 25.0 || 50.0  
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O   || 22.5  || 8.7
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| &nbsp;             || 50.0 μL || 
|}
|}


Thermal cycler: Labnet - GOTAQ
* 95°C 3 min.
* 30x[95°C, 30 sec; 57°C 30 sec; 72°C 30 sec]
* 72°C 3 min.
* 4°C, ∞


* 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 00:56, 27 September 2017

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

04/28/15

  • Ryan - minipreps & digests
  • Ryan - PCR & Dig/Lig (promoter insert trial #2)


Minipreps

  • Use Sigma GenElute; elute with 75 μL elution solution
  • BraR/MRV colonies - picked 4 additional colonies from original cloning plate
  • Cut w/ E/X
  1. Bra/MRV 3
  2. Bra/MRV 4
  3. Bra/MRV 5
  4. Bra/MRV 6
  • promoter/Regulator/MRV colonies
  • Cut w/ KpnI/EcoRI - BbsI drop-in of promoter destroys KpnI site; empty vector will produce 2 bands, successful clones will be linearized (1 cut)
  1. pAub/AubR/MRV
  2. pBja/BjaR/MRV
  3. pRpa/RpaR/MRV


Reagent Volume Expected:
1-4. Bra/MRV = ~3200, 776
Empty MRV E/X = ~3200
5. pAub/AubR/MRV = 4240
6. pBja/BjaR/MRV = 4099
7. pRpa/RpaR/MRV = 4116
Empty vector = ~4000, 971 		Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
enzyme 1 1.0
enzyme 2 1.0
dH2O 8.5
  15 μL --> 37°C/ ~15 min.


  • Measure [DNA]
Sample OD 260 260/280 ng/μL
BraR/MRV #5 0.465 1.913 465.4


Conclusions

  • BraR/MRV - Success! Keep colony 5 (lane 3)
  • promter/Reg/MRV - none worked. Try the assembly again with PCR-amplified promoter inserts


Ryan - PCR & Dig/Lig (promoter insert trial #2)

Ryan - Receiver plasmids, assembly - REVISED STRATEGY (PCR promoters)

  • 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
    • PCR-amplify promoter inserts (same primers as Stage 1)
    • Cut & dephos promoters with E/X
    • Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


Assemblies

  1. pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087
  2. pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977
  3. pBra/BraR/MRV: pBra(E/Xdp)/214 + BraR/MRV(BbsI)/3977
  4. pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980


  • Measure conc.'s
    • Values from last week, except BraR/MRV, which was measured today
Sample OD260 260/280 ng/μL
1. AubR/MRV 0.258 1.906 257.7
2. BjaR/MRV 0.177 1.904 177.3
3. BraR/MRV #5 0.465 1.913 465.4
4. RpaR/MRV 0.246 1.896 246.2
  • PCR-amplify promoters
    • 2x 50 μL rxns each
  1. pAubR, EcoRI fwd, XbaI rev
  2. pBjaR, EcoRI fwd, XbaI rev
  3. pBraR, EcoRI fwd, XbaI rev
  4. pRpaR, EcoRI fwd, XbaI rev
Reagent Vol Mix (x2) Expected:
1. pAubR = 153
2. pBjaR = 122
3. pBraR = 214
4. pRpaR = 136 		Hover name
5 μL/lane; 1% agarose; Ladder
gBlock DNA (2 ng/μL) 0.5 1.0
10 μM XbaI rev 1.0 2.0
10 μM EcoRI fwd 1.0 2.0
2x GoTaq green 25.0 50.0
dH2O 22.5 8.7
  50.0 μL

Thermal cycler: Labnet - GOTAQ

  • 95°C 3 min.
  • 30x[95°C, 30 sec; 57°C 30 sec; 72°C 30 sec]
  • 72°C 3 min.
  • 4°C, ∞




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