User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/28: 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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<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* David Tze - assemblies: linkers + hPCD
* David Tze - assemblies: linkers + hPCD
* Ben - DBN007 5 mL cultures




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| bgcolor=#cfcfcf | Rxn1
| bgcolor=#cfcfcf | Rxn1
| bgcolor=#cfcfcf | Rxn2-5
| bgcolor=#cfcfcf | Rxn2-5
| rowspan="7" | Expected bands:<br>1. hPCD(E/S) = 186<br>2. PLflex_pUC57(E/X) = 2763<br>3. PLflex4_pUC57(E/X) = 2930<br>4. PLrigid_pUC57(E/X) = 2763<br>5. PLrigid4_pUC57(E/X) = 2931
| rowspan="7" | Expected bands:<br>1. hPCD(E/S) = 3200, 186*<br>2. PLflex_pUC57(E/X) = 2763*<br>3. PLflex4_pUC57(E/X) = 2930*<br>4. PLrigid_pUC57(E/X) = 2763*<br>5. PLrigid4_pUC57(E/X) = 2931*<br><br>(*) Bands that were cut out for purification
| rowspan="7" | [[Image:somegel.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:KAH052815_gel1.jpg|250px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA (plasmid) || 25.0 || 15.0
| DNA (plasmid) || 25.0 || 15.0
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| 10x buffer || 3.0 || 3.0
| 10x buffer || 3.0 || 3.0
|-
|-
| enzyme 1 || 1.0 || 1.0
| EcoRI || 1.0 || 1.0
|-
|-
| enzyme 2 || 1.0 || 1.0
| enzyme 2 || 1.0 || 1.0
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|-
|-
| 2. PLflex_pUC57(E/X) || --- || --- || ---
| 2. PLflex_pUC57(E/X) || --- || --- || ---
|-
| 3. PLflex4_pUC57(E/X) || --- || --- || ---
|-
| 4. PLrigid_pUC57(E/X) || --- || --- || ---
|-
| 5. PLrigid4_pUC57(E/X) || --- || --- || ---
|}
|}
** Readings are very odd, which is typical for the Sigma gel extraction products.
** Will try my luck with these and use an arbitrary volume of 2.0 insert and 2.0 vector.
** Good insert : vector ratio should not be difficult to achieve since insert is very short.






* Ligations
* Ligations
# insert/(a/b)/size + vector/(c/d)/size
** 2:1 ratio calculation: 186 bp insert / ~2800 bp vector * 2 * 50 = 6.64 ng insert
# vector/(c/d)/size
# DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
# DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
# DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
# DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931
# PLflex_pUC57(E/X)/2763
* Note: not enough plates to do neg. ctrls for all vectors. Used just one of the vectors


{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1
| bgcolor=#cfcfcf | Rxn1-4
| bgcolor=#cfcfcf | Rxn2
| bgcolor=#cfcfcf | Rxn5
|-
|-
| Insert DNA        || ### || ---  
| Insert DNA        || 2.0 || ---  
|-
|-
| Vector DNA        || ### || ###
| Vector DNA        || 2.0 || 2.0
|-
|-
| 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    || --- || 2.0
|-
|-
| &nbsp;            || # μL || # μL
| &nbsp;            || 10.0 μL || 10.0 μL
|}
|}
RESULTS
* Looks successful - David Tze: ~200 colonies on the ligation plates, 1 colony on the neg. ctrl plate.
* David Tze: picked two colonies from each plate (1-4) for streak library and 5 mL cultures
----
'''Ben - DBN007 5 mL cultures'''
* DBN001_psB1A3: https://benchling.com/s/S2BS6ziD/edit
* DBN006: https://benchling.com/s/XoQEXLOz/edit
* Minipreps<br>
** Sigma GenElute kit, elute w/ 75 μL elution sln.
* Digests
** Cut w/ E/S
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1-2. DBN007_pSB1A3 = 2197, 2132<br>DBN001 = 2132, 224<br>
| rowspan="7" | [[Image:KAH052815_gel2.jpg|150px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| DNA(plasmid) || 2.0 μL
|-
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| SpeI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &nbsp; || 15 μL
|}
--> 37°C/ ~15 min.
CONCLUSIONS
* Looks successful. 2197, 2132 bands are hard to resolve at the 2000 kb range.
* Submit the plasmids for sequencing




Latest revision as of 00:59, 27 September 2017

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

05/28/15

  • David Tze - assemblies: linkers + hPCD
  • Ben - DBN007 5 mL cultures


David Tze - assemblies: linkers + hPCD

  • Assemblies
  1. DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
  2. DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
  3. DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
  4. DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931


  • Digests (Fermentas FD)
Reagent Rxn1 Rxn2-5 Expected bands:
1. hPCD(E/S) = 3200, 186*
2. PLflex_pUC57(E/X) = 2763*
3. PLflex4_pUC57(E/X) = 2930*
4. PLrigid_pUC57(E/X) = 2763*
5. PLrigid4_pUC57(E/X) = 2931*

(*) Bands that were cut out for purification 		Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 25.0 15.0
10x buffer 3.0 3.0
EcoRI 1.0 1.0
enzyme 2 1.0 1.0
dH2O --- 10.0
  30 μL

--> 37°C/ ~30 min.


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


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. hPCD(E/S) --- --- ---
2. PLflex_pUC57(E/X) --- --- ---
3. PLflex4_pUC57(E/X) --- --- ---
4. PLrigid_pUC57(E/X) --- --- ---
5. PLrigid4_pUC57(E/X) --- --- ---
    • Readings are very odd, which is typical for the Sigma gel extraction products.
    • Will try my luck with these and use an arbitrary volume of 2.0 insert and 2.0 vector.
    • Good insert : vector ratio should not be difficult to achieve since insert is very short.


  • Ligations
    • 2:1 ratio calculation: 186 bp insert / ~2800 bp vector * 2 * 50 = 6.64 ng insert
  1. DT001_pUC57: hPCD(E/S)/186 + PLflex_pUC57(E/X)/2763
  2. DT002_pUC57: hPCD(E/S)/186 + PLflex4_pUC57(E/X)/2930
  3. DT003_pUC57: hPCD(E/S)/186 + PLrigid_pUC57(E/X)/2763
  4. DT004_pUC57: hPCD(E/S)/186 + PLrigid4_pUC57(E/X)/2931
  5. PLflex_pUC57(E/X)/2763
  • Note: not enough plates to do neg. ctrls for all vectors. Used just one of the vectors
Reagent Rxn1-4 Rxn5
Insert DNA 2.0 ---
Vector DNA 2.0 2.0
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 2.0
  10.0 μL 10.0 μL


RESULTS

  • Looks successful - David Tze: ~200 colonies on the ligation plates, 1 colony on the neg. ctrl plate.
  • David Tze: picked two colonies from each plate (1-4) for streak library and 5 mL cultures


Ben - DBN007 5 mL cultures


  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.


  • Digests
    • Cut w/ E/S
Reagent Volume Expected:
1-2. DBN007_pSB1A3 = 2197, 2132
DBN001 = 2132, 224
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
SpeI 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~15 min.


CONCLUSIONS

  • Looks successful. 2197, 2132 bands are hard to resolve at the 2000 kb range.
  • Submit the plasmids for sequencing




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