User:Karmella Haynes/Notebook/BioBrick cloning/2015/06/23: Difference between revisions

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(Autocreate 2015/06/23 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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----
----
'''Line item'''
'''Assemblies'''
# H3-1-40_MV11: H3-1-40/(X/N)/129 + 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 + "


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


* Digests
* Digest of vector (Fermentas FD)
** Check with #/# digests
** SpeI/NotI


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} cellspacing="3" <!-- Digest rxn. 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>
| 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|400px|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) || 15.0
|-
|-
| 10X buffer || 1.5
| 10x buffer || 3.0
|-
|-
| EcoRI || 1.0
| SpeI || 1.0
|-
|-
| PstI || 1.0
| NotI || 1.0
|-
|-
| dH<sub>2</sub>O || 9.5
| dH<sub>2</sub>O || 10.0
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 30 μL --> 37°C/ ~15 min.
|}
|}


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




* 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


* 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.


* Digests (Fermentas FD)
** Specific notes


{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
* Measure [DNA] for vector and inserts
|- valign="top"
{| {{table}} cellspacing="3" <!-- [DNA] table -->
| bgcolor=#cfcfcf | Reagent
|- bgcolor=#cfcfcf  
| bgcolor=#cfcfcf | Volume
| Sample || OD260 || 260/280 || ng/μL
| 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
| 1. MV11 (S/N) || 0.03 || 1.879 || 13.5
|-
|-
| 10x buffer || 3.0
| 2. H3-1-40 PCR || 0.03 || 1.879 || 29.8
|-
|-
| enzyme 1 || 1.0
| 3. H3-1-60 PCR || 0.04 || 1.974 || 39.7
|-
|-
| enzyme 2 || 1.0
| 4. H3-1-80 PCR || 0.047 || 1.865 || 46.8
|-
|-
| dH<sub>2</sub>O || ---
| 5. H3-1-116 PCR || 0.06 || 1.887 || 59.57
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| 6. H3-1-136 PCR || 0.062 || 1.932 || 62.0
|}
|}




* Measure conc.'s
* Digest & dephos inserts
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. Digested part (a/b) || --- || --- || ---
|-
| 2. Digested part (c/d) || --- || --- || ---
|}


 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
* Dephosphorylation (Roche)
|- valign="top"
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| 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
# insert/(a/b)/size + vector/(c/d)/size
** > 2:1 ratio calculations...
# vector/(c/d)/size
** 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


{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
|- valign="top"
|- bgcolor="#cfcfcf" valign="top"
| bgcolor=#cfcfcf | Reagent
| Reagent
| bgcolor=#cfcfcf | Rxn1
| Rxn1-5
| bgcolor=#cfcfcf | Rxn2
| 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




Revision as of 16:32, 23 June 2015

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mm/dd/yy

  • Line item 1
  • Line item 2


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



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