User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/17

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(Autocreate 2013/01/17 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
Current revision (16:06, 3 March 2013) (view source)
(→01/17/13)
 
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==mm/dd/yy==
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==01/17/13==
<!-- Precede finished items with a checkmark &#x2713; -->
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* Line item 1
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* Minipreps: check Golden Gate assembly clones (trial 3)
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* Line item 2
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'''Minipreps'''<br>
'''Minipreps'''<br>
* Check with E/P digests
* Check with E/P digests
 +
* Minipreps from colonies on Plate #2 (Promega home-made ligation buffer trial)
{| {{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  
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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" | <u>Expected:</u><br>1. Plate 2-1 = 2000 (v), 2706 (i)<br>2. Plate 2-2 = 2000 (v), 2706 (i)<br>3. Plate 2-3 (red) = 2000 (v), 2706 (i)<br><br>4. Plate 2-4 = 2000 (v), 2706 (i)<br>5. pSB1A3+RFP = 2000 (v), ~1200 (i)<br>6. hPCD/V0120 = 3200 (v), 186 (i)<br>7. BL01 = 3200 (v), 2520 (i)<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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| rowspan="7" | [[Image:KAH011813_gel1.jpg|300px|E/P digests]]<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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| DNA(plasmid) || 3.0 μL
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| 10X buffer || 1.5
| 10X buffer || 1.5
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|}
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----
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Conclusions
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'''Assemblies'''
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* '''Success!''' (1, 2, & 4) Bands match expected size for vector pSB1A3 (not old vector V0120) and for assembled insert: hPCD (186) + BL01 (2520) = 2706 (visibly larger than BL01 band)
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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* Transferring inserts from V0120 to pSB1A3 is probably a good idea since several internal BsmBI sites might destroy any template background
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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* Use BsmBI Type IIS method to assemble all the things!
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* Digests (Fermentas FD)
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1/23/13 Sequencing confirmation (DNASU)
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** Specific notes
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# ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01
 +
# ggPro-1, VR - correct sequence for SP1AB of BL01
 +
# ggPro-2, VF2 - matches ggPro-1 forward (BLAST)
 +
# ggPro-2, VR - matches ggPro-1 reverse (BLAST)
 +
# ggPro-4, VF2 - matches ggPro-1 forward (BLAST)
 +
#ggPro-4, VR - matches ggPro-1 reverse (BLAST)
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{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
 
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|- valign="top"
 
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| bgcolor=#cfcfcf | Reagent
 
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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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|-
 
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| 10x buffer || 3.0
 
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|-
 
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| enzyme 1 || 1.0
 
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|-
 
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| enzyme 2 || 1.0
 
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|-
 
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| dH<sub>2</sub>O || ---
 
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|-
 
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| &nbsp; || 30 μL --> 37°C/ ~30 min.
 
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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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|-
 
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| 2. Digested part (c/d) || --- || --- || ---
 
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|}
 
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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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|-
 
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| 10x buffer d.p. || 2.0
 
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|-
 
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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
 
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|}
 
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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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|}
 
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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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|}
 
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----
 
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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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|}
 

Current revision

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

01/17/13

  • Minipreps: check Golden Gate assembly clones (trial 3)


Minipreps

  • Check with E/P digests
  • Minipreps from colonies on Plate #2 (Promega home-made ligation buffer trial)
Reagent Volume Expected:
1. Plate 2-1 = 2000 (v), 2706 (i)
2. Plate 2-2 = 2000 (v), 2706 (i)
3. Plate 2-3 (red) = 2000 (v), 2706 (i)

4. Plate 2-4 = 2000 (v), 2706 (i)
5. pSB1A3+RFP = 2000 (v), ~1200 (i)
6. hPCD/V0120 = 3200 (v), 186 (i)
7. BL01 = 3200 (v), 2520 (i)
 E/P digests
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Conclusions

  • Success! (1, 2, & 4) Bands match expected size for vector pSB1A3 (not old vector V0120) and for assembled insert: hPCD (186) + BL01 (2520) = 2706 (visibly larger than BL01 band)
  • Transferring inserts from V0120 to pSB1A3 is probably a good idea since several internal BsmBI sites might destroy any template background
  • Use BsmBI Type IIS method to assemble all the things!


1/23/13 Sequencing confirmation (DNASU)

  1. ggPro-1, VF2 - correct sequence, scarless junction between hPCD and BL01
  2. ggPro-1, VR - correct sequence for SP1AB of BL01
  3. ggPro-2, VF2 - matches ggPro-1 forward (BLAST)
  4. ggPro-2, VR - matches ggPro-1 reverse (BLAST)
  5. ggPro-4, VF2 - matches ggPro-1 forward (BLAST)
  6. ggPro-4, VR - matches ggPro-1 reverse (BLAST)



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