User:Karmella Haynes/Notebook/BioBrick cloning/2013/09/06: Difference between revisions

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==mm/dd/yy==
==09/06/13==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Line item 1
* Miniprep - CMV
* Line item 2
* Restriction digest - CMV
* Sequencing - CMV [cloning bootcamp demo]




----
'''Minipreps'''<br>
* Check with E/P digests
{| {{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.
|}


----
----
'''Assemblies'''
'''Results'''
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
* CMV/V0120
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
** Culture innoculated on [http://openwetware.org/Karmella_Haynes/Notebook/BioBrick_cloning/2013/09/05 9/05/13]
** Results for liquid cultures: (1) grew, (2) grew
** Results for streak plate: (1) grew well, (2) hardly any growth




* Digests (Fermentas FD)
'''List of Samples for Minprep & Analysis'''
** Specific notes
# Sample 1 = CMV/V0120 colony 1; BBa_J176027; part = 588 bp; vector = 3200
# Sample 2 = CMV/V0120 colony 2; same
# Sample 3 = CMV/V0120 (control DNA); BBa_J176027; part = 588 bp; vector = 3200


{| {{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.
|}


'''DNA Concentration Data'''
<!-- Wiki code notes: The {| is the opening tag for tables. {{table}} is an OWW template that creates thin grey border lines and cell margins.
<!-- Each |- starts a new row. There are three rows. bgcolor=#cfcfcf *colors* the *background* of the first row grey.  -->
<!-- Below that, each | starts a row of cells. -->
<!-- The symbol || separates cells in a row. Replace the --- in each cell with your data. -->
<!-- If you have only one plasmid, delete the |- under Plasmid 1's row, and delete the entire row for Plasmid 2.-->
<!-- |} must be the last symbol (on its own line) in the table. This is the table closing tag. -->


* Measure conc.'s
{| {{table}}  
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf  
|- bgcolor=#cfcfcf  
| Sample || OD260 || 260/280 || ng/μL
| Plasmid || OD260 || OD260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 1. Plasmid 1 || 0.108 || 1.841 || 108.321
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| 2. Plasmid 2 || 0.007 || 1.75 || 6.60
|}
|}
* Streak #2 did not grow well. Culture might be empty bacteria. Use mini prep 1 for other steps.




* Dephosphorylation (Roche)
'''Restriction Digest Table'''<br>
{| {{table}} cellspacing="3" <!-- Dephos table -->
* Checked plasmid minipreps with EcoRI/PstI digests
|-
 
| bgcolor=#cfcfcf | Reagent
{| {{table}} border="1" cellspacing="3"  
<!-- Editing: the coding for this table is a bit more advanced. -->
<!-- valign="top" aligns all the text in the first row to the top.  -->
<!-- The | symbols on the next two lines start new cells in the same row. This does the same thing as ||, but you have to use | on new lines to set formatting for cells. -->
<!-- bgcolor=#cfcfcf *colors* the *background* of the Reagent and Volume cells grey.  -->
<!-- The next two "rowspan=7" cells span  all 7 rows in the table so that they can fit the "Expected" list and a gel image. rowspan is how you create merged rows in Wiki code. -->
<!-- Replace Plasmid 1 and 2 with the names of your plasmids. Replace insert size and vector size with appropriate information for your plasmids. If you only have one Plasmid, delete all the text for Plasmid 2
<!-- After you upload your gel image to OWW, replace "GelImage.jpg" with the name of your image file -->
<!-- &nbsp; is ASCII code for an invisible space. -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent  
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Volume
| rowspan="7" | <u>Expected:</u><br>1. CMV colony 1 = 588, 3200<br>2. CMV colony 2 = 588, 3200<br>3. CMV control = 588, 3200<br>
| rowspan="7" | [[Image:GelImage.jpg|400px|Today's gel]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA (clean digest) || up to 17 μL (500 ng)
| DNA(plasmid) || 3.0 μL
|-
|-
| 10x buffer d.p. || 2.0
| 10X buffer || 1.5
|-
|-
| phosphatase || 1.0
| EcoRI || 1.0
|-
|-
| dH<sub>2</sub>O || ---
| PstI || 1.0
|-
| &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) || ###  || ###  ||
| dH<sub>2</sub>O || 8.5
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| &nbsp; || 15 μL --> 37°C/ 15 min.
|-
| 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 Sequencing Samples'''
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
<!-- * signs create an automatically bulleted list. Replace 'date' with the date you submitted the samples -->
|-
* Submitted to DNASU on 'date'
| 10x annealing buffer || 2.0
<!-- # signs create an automatically numbered list. Replace Plasmid 1 & 2 with the names of your plasmids. Replace 'name' with the appropriate name of each primer. -->
|-
# Plasmid 1 - forward primer 'name'
| dH<sub>2</sub>O || ---
# Plasmid 1 - reverse primer 'name'
|-
# Plasmid 2 - forward primer 'name'
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
# Plasmid 2 - reverse primer 'name'
|}




Revision as of 19:25, 6 September 2013

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09/06/13

  • Miniprep - CMV
  • Restriction digest - CMV
  • Sequencing - CMV [cloning bootcamp demo]


Results

  • CMV/V0120
    • Culture innoculated on 9/05/13
    • Results for liquid cultures: (1) grew, (2) grew
    • Results for streak plate: (1) grew well, (2) hardly any growth


List of Samples for Minprep & Analysis

  1. Sample 1 = CMV/V0120 colony 1; BBa_J176027; part = 588 bp; vector = 3200
  2. Sample 2 = CMV/V0120 colony 2; same
  3. Sample 3 = CMV/V0120 (control DNA); BBa_J176027; part = 588 bp; vector = 3200


DNA Concentration Data

Plasmid OD260 OD260/280 ng/μL
1. Plasmid 1 0.108 1.841 108.321
2. Plasmid 2 0.007 1.75 6.60
  • Streak #2 did not grow well. Culture might be empty bacteria. Use mini prep 1 for other steps.


Restriction Digest Table

  • Checked plasmid minipreps with EcoRI/PstI digests
Reagent Volume Expected:
1. CMV colony 1 = 588, 3200
2. CMV colony 2 = 588, 3200
3. CMV control = 588, 3200
 Today's gel
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 8.5
  15 μL --> 37°C/ 15 min.


DNA Sequencing Samples

  • Submitted to DNASU on 'date'
  1. Plasmid 1 - forward primer 'name'
  2. Plasmid 1 - reverse primer 'name'
  3. Plasmid 2 - forward primer 'name'
  4. Plasmid 2 - reverse primer 'name'



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