User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/28: Difference between revisions

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(Autocreate 2015/04/28 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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==mm/dd/yy==
==04/28/15==
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* Line item 1
* Ryan - minirpeps & digests
* Line item 2
* Line item 2


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----
----
'''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 = ###<br>Empty MRV E/X = ###<br>5. pAub/AubR/MRV = ###<br>6. pBja/BjaR/MRV = ###<br>7. pRpa/RpaR/MRV = ###<br>Empty vector = ~###, 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: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) || 3.0 μL
|-
|-
| 10X buffer || 1.5
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}
----
'''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
* Digests (Fermentas FD)
** Specific notes
{| {{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 1 || 1.0
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| enzyme 2 || 1.0
| enzyme 2 || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || 8.5
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 15 μL --> 37°C/ ~15 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
{| {{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
|}




Revision as of 12:56, 28 April 2015

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04/28/15

  • Ryan - minirpeps & digests
  • Line item 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 = ###
Empty MRV E/X = ###
5. pAub/AubR/MRV = ###
6. pBja/BjaR/MRV = ###
7. pRpa/RpaR/MRV = ###
Empty vector = ~###, 971
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.



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