User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/22: 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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* Ryan - Bra minipreps & digests
* Ryan - Bra minipreps & digests
* Cas-tone - minipreps & digests
* Cas-tone - minipreps & digests
* Ben - luc replacement donor plasmid




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| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1-8. pBra-BraR_MRV = 2989, 1202<br>empty vector = 3977<br>
| rowspan="7" | <u>Expected:</u><br>1-8. pBra-BraR_MRV = 2989, 1202<br>empty vector = 3977<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] -->
| rowspan="7" | [[Image:KAH052215_gel1.jpg|250px|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) || 2.0 μL
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** Make streak library & 5 mL cultures
** Make streak library & 5 mL cultures


 
* Digests
** Check with S/HindIII digests - H3 insertion destroys SpeI site
** Check with S/HindIII digests - H3 insertion destroys SpeI site
** Note: Fragment without H3 part = 7162; added H3-1-# size + 7162 to get lengths for longer H/H band
** Note: Fragment without H3 part = 7162; added H3-1-# size + 7162 to get lengths for longer H/H band
** H3-1-40 = 129; H3-1-60 = 189; H3-1-80 = 250; H3-1-116 = 373; H3-1-136 = 433


{| {{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>1,2. H3-1-40_MV11 = 2345, 7161+##<br>3,4. H3-1-60_MV11 = 2345, 7162+##<br>5,6. H3-1-80_MV11 = 2345, 7162+##<br>7,8. H3-1-116_MV11 = 2345, 7162+##<br>9,10. H3-1-136_MV11 = 2345, 7162+##<br>
| rowspan="7" | <u>Expected:</u><br>1,2. H3-1-40_MV11 = 2345, 7291<br>3,4. H3-1-60_MV11 = 2345, 7351<br>5,6. H3-1-80_MV11 = 2345, 7412<br>7,8. H3-1-116_MV11 = 2345, 7535<br>9,10. H3-1-136_MV11 = 2345, 7595<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] -->
| rowspan="7" | [[Image:KAH052215_gel2.jpg|250px|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) || 2.0 μL
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----
'''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 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
{| {{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
|}




Latest revision as of 00:59, 27 September 2017

Karmella's BioBrick Cloning Main project page
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05/22/15

  • Ryan - Bra minipreps & digests
  • Cas-tone - minipreps & digests


Ryan - Bra minipreps & digests

  • Minipreps
    • Sigma GenElute kit, elute w/ 75 μL elution sln.
  • Digests
    • Check with EcoRI digests - promoter insertion generates a new EcoRI site
    • Note: BraR = 776, pBra = 214
Reagent Volume Expected:
1-8. pBra-BraR_MRV = 2989, 1202
empty vector = 3977
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
dH2O 10.5
  15 μL

--> 37°C/ ~15 min.


Cas-tone - H3_MV11 minipreps & digests

  • Cultures
    • Pick 2 colonies from each ligation plate
    • Make streak library & 5 mL cultures
  • Digests
    • Check with S/HindIII digests - H3 insertion destroys SpeI site
    • Note: Fragment without H3 part = 7162; added H3-1-# size + 7162 to get lengths for longer H/H band
    • H3-1-40 = 129; H3-1-60 = 189; H3-1-80 = 250; H3-1-116 = 373; H3-1-136 = 433
Reagent Volume Expected:
1,2. H3-1-40_MV11 = 2345, 7291
3,4. H3-1-60_MV11 = 2345, 7351
5,6. H3-1-80_MV11 = 2345, 7412
7,8. H3-1-116_MV11 = 2345, 7535
9,10. H3-1-136_MV11 = 2345, 7595
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
SpeI 1.0
HindIII 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~15 min.




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