User:Karmella Haynes/Notebook/BioBrick cloning/2013/08/29: Difference between revisions

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==mm/dd/yy==
==08/29/13==
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* Line item 1
* Golden Gate Assembly optimization - increase ligase amount; redo last successful hPCD assembly
* Line item 2




----
----
'''Minipreps'''<br>
'''Golden Gate'''
* Check with E/P digests
* Try to increase robustness by using more T4 ligase...
# hPCD + BL01 + pSB1A3
# pSB1A3 alone (neg ctrl)
* GG-PCR fragments were generated on [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/01/10 1/10/13] and diluted to 20 fmol/μL
* Try the Promega formula home-made buffer (worked last time)


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| 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
|-
| 10X buffer || 1.5
|-
| EcoRI || 1.0
|-
| PstI || 1.0
|-
| dH<sub>2</sub>O || 9.5
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
|}


----
* Golden Gate assembly reactions
'''Assemblies'''
1, 2. 0.25 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3<br>
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
1, 2. 1.0 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3<br>
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size




* Digests (Fermentas FD)
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
** Specific notes
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| Reagent || Original protocol  || Original protocol || More ligase || More ligase || H2B+LOV
| 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) || --- || --- || ---
| gg2 pSB1A3* || 1.0 || 1.0 || 1.0 || 1.0 || 1.0
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| gg3 hPCD* || 1.0 || --- || 1.0 || --- || 1.0 H2B gg
|}
 
 
* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
|-
| bgcolor=#cfcfcf | Reagent
| gg4 BL01* || 1.0 || --- || 1.0 || --- || 1.0 LOV gg
| bgcolor=#cfcfcf | Volume
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| 10x Promega ligase buffer || 1.0 || 1.0|| 1.0 || 1.0 || 1.0
|-
|-
| 10x buffer d.p. || 2.0
| NEB T4 ligase || 0.25 || 0.25 || 1.0 || 1.0 || 1.0
|-
|-
| phosphatase || 1.0
| NEB BsmBI || 0.5 || 0.5 || 0.5 || 0.5 || 0.5
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || 5.25 || 7.25 || 4.5 || 6.5 || 4.5
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| &nbsp; || 10.0 || 10.0 || 10.0 || 10.0 || 10.0
|}
|}
Note: *DNA is 20 fmol/μL


GG Thermal cycling
* [45°C, 2 min.; 16°C, 5 min.] x25
* 60°C, 20 min.
* 80°C, 20 min.
* 4°C, ∞


* 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 -->
* Transformations (08/30/13)
| &nbsp;            || 1    || 2    ||
** 40 μL '''BL21''' in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 750 μL SOC medium; shake @ 37°C 30 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar
|-
| Insert DNA        || ###  || ---  ||
|-
| Vector DNA        || ###  || ###  ||
|-
| 2x lgn buf (Roche) || ###  || ###  ||
|-
| T4 ligase (NEB)    || 1.0 || 1.0  ||
|-
| dH<sub>2</sub>O    || ###  || ###  ||
|-
| &nbsp;             || # μL || # μL ||
|}


----
* Results
'''Oligo annealing'''
** No colonies on any plates. Start again from generation of PCR fragments. Try DpnI clean-up approach next time.
# 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 12:09, 31 August 2013

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08/29/13

  • Golden Gate Assembly optimization - increase ligase amount; redo last successful hPCD assembly


Golden Gate

  • Try to increase robustness by using more T4 ligase...
  1. hPCD + BL01 + pSB1A3
  2. pSB1A3 alone (neg ctrl)
  • GG-PCR fragments were generated on 1/10/13 and diluted to 20 fmol/μL
  • Try the Promega formula home-made buffer (worked last time)


  • Golden Gate assembly reactions

1, 2. 0.25 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3
1, 2. 1.0 μL T4 ligase, hPCD+BL01+pSB1A3 or pSB1A3


Reagent Original protocol Original protocol More ligase More ligase H2B+LOV
gg2 pSB1A3* 1.0 1.0 1.0 1.0 1.0
gg3 hPCD* 1.0 --- 1.0 --- 1.0 H2B gg
gg4 BL01* 1.0 --- 1.0 --- 1.0 LOV gg
10x Promega ligase buffer 1.0 1.0 1.0 1.0 1.0
NEB T4 ligase 0.25 0.25 1.0 1.0 1.0
NEB BsmBI 0.5 0.5 0.5 0.5 0.5
dH2O 5.25 7.25 4.5 6.5 4.5
  10.0 10.0 10.0 10.0 10.0

Note: *DNA is 20 fmol/μL

GG Thermal cycling

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


  • Transformations (08/30/13)
    • 40 μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 750 μL SOC medium; shake @ 37°C 30 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar
  • Results
    • No colonies on any plates. Start again from generation of PCR fragments. Try DpnI clean-up approach next time.



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