User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/29: 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==
==05/29/15==
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* David Tze - minpreps & digests
* David Tze - minpreps & digests
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| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1. DT001 = size<br>2. DT002 = size<br>3. DT003 = size<br>4. DT004 = size<br>Empty vectors = ~2600, ~400 - 600
| rowspan="7" | <u>Expected:</u><br>1. DT001 = 2668, 252<br>Empty PLflex = 2668, 95<br>2. DT002 = 2656, 432<br>Empty PLflex4 = 2656, 274<br>3. DT003 = 2668, 252<br>Empty PLrigid = 2668, 95<br>4. DT004 = 2656, 432<br>Empty PLrigid4 = 2656, 275
| 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:KAH052915_gel1.jpg|250px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| DNA(plasmid) || 2.0 μL
| DNA(plasmid) || 2.0 μL
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----
CONCLUSIONS:
'''Sequencing orders'''
# DT001 - success.
# DT002 - success.
# DT003 - success.
# DT004 - success.


# DBN007_1 -


* Next steps:
** Sequence verify all clones (extra precaution; linker part is small)


* Assemblies
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


----
'''Sequencing order'''
* 1-4 - Ben
* 5-16 - Cas-tone


* Digests (Fermentas FD)
# DBN007-1 - VF2 - good
** Specific notes
# DBN007-1 - VR - good, pSB1A3 terminator appears to be deleted
# DBN007-2 - VF2 - good
# DBN007-2 - VR  - good, pSB1A3 terminator appears to be deleted
# H3140-1 - CMVf
# H3140-1 - DD123
# H3140-2 - CMVf
# H3140-2 - DD123
# H3160-1 - CMVf
# H3160-1 - DD123
# H3160-2 - CMVf
# H3160-2 - DD123
# H3180-1 - CMVf
# H3180-1 - DD123
# H3180-2 - CMVf
# H3180-2 - DD123
# H31116-2 - CMVf
# H31116-2 - DD123
# H31136-1 - CMVf
# H31136-1 - DD123


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| 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] -->
|-
| 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
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. Digested part (a/b) || --- || --- || ---
|-
| 2. Digested part (c/d) || --- || --- || ---
|}
* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| 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
|}
* Ligations
# insert/(a/b)/size + vector/(c/d)/size
# vector/(c/d)/size
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1
| bgcolor=#cfcfcf | Rxn2
|-
| Insert DNA        || ###  || ---
|-
| Vector DNA        || ###  || ###
|-
| 2x lgn buf (Roche) || ###  || ###
|-
| T4 ligase (NEB)    || 1.0  || 1.0
|-
| dH<sub>2</sub>O    || ###  || ###
|-
| &nbsp;            || # μL || # μL
|}




Latest revision as of 01:00, 27 September 2017

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

05/29/15

  • David Tze - minpreps & digests
  • Ben - sequencing order
  • Cas-tone - sequencing order


David Tze - minpreps & digests

  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  • Digests
    • Check with E/S digests; cannot use PstI because PLrigid & PLrigid4 contain PstI sites
Reagent Volume Expected:
1. DT001 = 2668, 252
Empty PLflex = 2668, 95
2. DT002 = 2656, 432
Empty PLflex4 = 2656, 274
3. DT003 = 2668, 252
Empty PLrigid = 2668, 95
4. DT004 = 2656, 432
Empty PLrigid4 = 2656, 275 		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:

  1. DT001 - success.
  2. DT002 - success.
  3. DT003 - success.
  4. DT004 - success.


  • Next steps:
    • Sequence verify all clones (extra precaution; linker part is small)


Sequencing order

  • 1-4 - Ben
  • 5-16 - Cas-tone
  1. DBN007-1 - VF2 - good
  2. DBN007-1 - VR - good, pSB1A3 terminator appears to be deleted
  3. DBN007-2 - VF2 - good
  4. DBN007-2 - VR - good, pSB1A3 terminator appears to be deleted
  5. H3140-1 - CMVf
  6. H3140-1 - DD123
  7. H3140-2 - CMVf
  8. H3140-2 - DD123
  9. H3160-1 - CMVf
  10. H3160-1 - DD123
  11. H3160-2 - CMVf
  12. H3160-2 - DD123
  13. H3180-1 - CMVf
  14. H3180-1 - DD123
  15. H3180-2 - CMVf
  16. H3180-2 - DD123
  17. H31116-2 - CMVf
  18. H31116-2 - DD123
  19. H31136-1 - CMVf
  20. H31136-1 - DD123





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