User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/15

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==mm/dd/yy==
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==01/15/13==
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* Line item 1
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* Golden gate assembly: test different ligase buffers
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* Line item 2
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----
----
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'''Minipreps'''<br>
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'''Golden Gate Assembly optimization'''<br>
-
* Check with E/P digests
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* Test different buffers
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
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|- valign="top"
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|- valign="top" bgcolor=#cfcfcf  
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| bgcolor=#cfcfcf | Reagent
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| Buffer || Tris-HCl || MgCl<sub>2</sub> || DTT || ATP
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| bgcolor=#cfcfcf | Volume
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| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
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-
| 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] -->
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|-
|-
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| DNA(plasmid) || 2.0 μL
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| 10x Promega || 300 mM (pH 7.8) || 100 mM || 100 mM || 10 mM
|-
|-
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| 10X buffer || 1.5
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| 10x NEB || 500 mM (pH 7.5) || 100 mM || 100 mM || 10 mM
|-
|-
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| EcoRI || 1.0
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| 2x Roche  || ? || ? || ? || ?
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|-
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| PstI || 1.0
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-
|-
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| dH<sub>2</sub>O || 9.5
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-
|-
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| &nbsp; || 15 μL --> 37°C/ ~15 min.
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|}
|}
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----
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* Make Promega buffer from scratch.
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'''Assemblies'''
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-
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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-
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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* Digests (Fermentas FD)
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* Golden Gate assembly reactions
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** Specific notes
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1, 2. Promega - hPCD+BL01+pSB1A3<br>
-
 
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3, 4. Promega - pSB1A3<br>
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{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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5, 6. NEB - hPCD+BL01+pSB1A3<br>
 +
7, 8. NEB - pSB1A3<br>
 +
9, 10. Roche - hPCD+BL01+pSB1A3<br>
 +
11, 12. Roche - pSB1A3<br>
 +
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
|- valign="top"
|- valign="top"
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| bgcolor=#cfcfcf | Reagent
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| Reagent || Promega (1,2)  || Promega (3, 4) || NEB (5, 6) || NEB (7, 8) || Roche (9, 10) || Roche (11, 12)
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| bgcolor=#cfcfcf | Volume
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| 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] -->
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|-
|-
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| DNA (plasmid) || up to 25 μL
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| gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0
|-
|-
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| 10x buffer || 3.0
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| gg3 hPCD* || 1.0 || --- || 1.0 || --- || 1.0 || ---
|-
|-
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| enzyme 1 || 1.0
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| gg4 BL01* || 1.0 || --- || 1.0 || --- || 1.0 || ---
|-
|-
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| enzyme 2 || 1.0
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| ligase buffer || 1.0 || 1.0|| 1.0 || 1.0|| 5.0 || 5.0
|-
|-
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| dH<sub>2</sub>O || ---
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| NEB T4 ligase || 0.25 || 0.25 || 0.25 || 0.25 || 0.25 || 0.25
|-
|-
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
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| NEB BsmBI || 0.5 || 0.5 || 0.5 || 0.5 || 0.5 || 0.5
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|}
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-
 
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-
 
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* Measure conc.'s
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{| {{table}} cellspacing="3" <!-- [DNA] table -->
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|- bgcolor=#cfcfcf
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| Sample || OD260 || 260/280 || ng/μL
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|-
|-
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| 1. Digested part (a/b) || --- || --- || ---
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| dH<sub>2</sub>O || 5.25 || 7.25 || 5.25 || 7.25 || 1.25 || 3.25
|-
|-
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| 2. Digested part (c/d) || --- || --- || ---
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| &nbsp; || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 || 10.0
|}
|}
 +
Note: *DNA is 20 fmole/μL
 +
Thermal cycler
 +
* [45°C, 2 min.; 16°C, 5 min.] x25
 +
* 60°C, 20 min.
 +
* 80°C, 20 min.
 +
* 4°C, ∞
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* Dephosphorylation (Roche)
 
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{| {{table}} cellspacing="3" <!-- Dephos table -->
 
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|-
 
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| bgcolor=#cfcfcf | Reagent
 
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| bgcolor=#cfcfcf | Volume
 
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|-
 
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| DNA (clean digest) || up to 17 μL (500 ng)
 
-
|-
 
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| 10x buffer d.p. || 2.0
 
-
|-
 
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| phosphatase || 1.0
 
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|-
 
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| dH<sub>2</sub>O || ---
 
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|-
 
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| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
 
-
|}
 
 +
* Transformations
 +
** All odd samples: 50 μL '''DH5α-Turbo'''; ice 5 min.; plate on amp agar
 +
** All even samples: 50 μL '''BL21''' in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar
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* Ligations
 
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{| {{table}} cellspacing="3" <!-- Ligations table -->
 
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|- bgcolor=#cfcfcf
 
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| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
 
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|-
 
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| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
 
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|-
 
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| 2. vector(c/d)/ ## ng || &nbsp;
 
-
|}
 
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{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
 
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| &nbsp;            || 1    || 2    ||
 
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|-
 
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| Insert DNA        || ###  || ---  ||
 
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|-
 
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| Vector DNA        || ###  || ###  ||
 
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|-
 
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| 2x lgn buf (Roche) || ###  || ###  ||
 
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|-
 
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| T4 ligase (NEB)    || 1.0  || 1.0  ||
 
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|-
 
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| dH<sub>2</sub>O    || ###  || ###  ||
 
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|-
 
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| &nbsp;            || # μL || # μL ||
 
-
|}
 
----
----
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'''Oligo annealing'''
 
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# New BB 1
 
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# New BB 2
 
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{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
 
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| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
 
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|-
 
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| 10x annealing buffer || 2.0
 
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|-
 
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| dH<sub>2</sub>O || ---
 
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|-
 
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| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
 
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|}
 
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<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Revision as of 21:12, 16 January 2013

Karmella's BioBrick Cloning Main project page
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01/15/13

  • Golden gate assembly: test different ligase buffers



Golden Gate Assembly optimization

  • Test different buffers
Buffer Tris-HCl MgCl2 DTT ATP
10x Promega 300 mM (pH 7.8) 100 mM 100 mM 10 mM
10x NEB 500 mM (pH 7.5) 100 mM 100 mM 10 mM
2x Roche  ?  ?  ?  ?
  • Make Promega buffer from scratch.


  • Golden Gate assembly reactions

1, 2. Promega - hPCD+BL01+pSB1A3
3, 4. Promega - pSB1A3
5, 6. NEB - hPCD+BL01+pSB1A3
7, 8. NEB - pSB1A3
9, 10. Roche - hPCD+BL01+pSB1A3
11, 12. Roche - pSB1A3

Reagent Promega (1,2) Promega (3, 4) NEB (5, 6) NEB (7, 8) Roche (9, 10) Roche (11, 12)
gg2 pSB1A3* 1.0 1.0 1.0 1.0 1.0 1.0
gg3 hPCD* 1.0 --- 1.0 --- 1.0 ---
gg4 BL01* 1.0 --- 1.0 --- 1.0 ---
ligase buffer 1.0 1.0 1.0 1.0 5.0 5.0
NEB T4 ligase 0.25 0.25 0.25 0.25 0.25 0.25
NEB BsmBI 0.5 0.5 0.5 0.5 0.5 0.5
dH2O 5.25 7.25 5.25 7.25 1.25 3.25
  10.0 10.0 10.0 10.0 10.0 10.0

Note: *DNA is 20 fmole/μL

Thermal cycler

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.
  • 4°C, ∞


  • Transformations
    • All odd samples: 50 μL DH5α-Turbo; ice 5 min.; plate on amp agar
    • All even samples: 50 μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar




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