User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/10: Difference between revisions

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(Autocreate 2015/04/10 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
 
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==mm/dd/yy==
==04/10/15==
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* Line item 1
* LCR development - Backbone PCR, Phusion and temp gradient, re-do
* Line item 2




----
'''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'''
'''LCR development - Backbone PCR'''<br>
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
* Repeat PCR from 04/09/15, use fresh dNTP's
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
* '''MV10''' (5191 bp): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1
* Gradient PCR to enhance 5 kb band
# 68°C
# 67.3°C
# 65.9°C
# 63.9°C
# 61.4°C
# 59.6°C
# 58.1°C
# 57°C


 
{| {{table}} cellspacing="3" <!-- PCR rxn. table -->
* Digests (Fermentas FD)
** Specific notes
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Rxn1-8
| 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] -->
| bgcolor=#cfcfcf | (x9)
| rowspan="7" | Expected:<br>1. Gal4DB-mCh = 1170<br>3,4. ATF2 = 906<br>5,6. MV10 = 5191
| rowspan="7" | [[Image:somegel.jpg|200px|Hover name]]<br>10 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA (plasmid) || up to 25 μL
| Template || 0.2 || 1.8
|-
|-
| 10x buffer || 3.0
| 10 uM fwd primer || 1.0 || 9.0
|-
|-
| enzyme 1 || 1.0
| 10 uM rev primer || 1.0 || 9.0
|-
|-
| enzyme 2 || 1.0
| 10 mM dNTPs || 1.0 || 9.0
|-
|-
| dH<sub>2</sub>O || ---
| Phusion pol. || 0.5 || 4.5
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| 5x HF buffer || 5.0 || 45.0
|}
 
 
* Measure conc.'s
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| dH<sub>2</sub>O || 41.3 || 371.7
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| &nbsp; || 50.0
|}
|}


Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient
* 98°C, 3 min
* 30x[98°C, 10 sec; 68°C - 57°C, 30 sec; 72°C, 2.5 min]
* 72°C, 10 min
* 4°C ∞


* 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 -->
Conclusions
| &nbsp;            || 1    || 2    ||
* tba
|-
| 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
|}




Revision as of 16:20, 10 April 2015

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04/10/15

  • LCR development - Backbone PCR, Phusion and temp gradient, re-do


LCR development - Backbone PCR

  • Repeat PCR from 04/09/15, use fresh dNTP's
  • MV10 (5191 bp): Template - MV10 plasmid, F - MV10 f1, R - MV10 r1
  • Gradient PCR to enhance 5 kb band
  1. 68°C
  2. 67.3°C
  3. 65.9°C
  4. 63.9°C
  5. 61.4°C
  6. 59.6°C
  7. 58.1°C
  8. 57°C
Reagent Rxn1-8 (x9) Expected:
1. Gal4DB-mCh = 1170
3,4. ATF2 = 906
5,6. MV10 = 5191 		Hover name
10 μL/lane, 1% agarose; Ladder
Template 0.2 1.8
10 uM fwd primer 1.0 9.0
10 uM rev primer 1.0 9.0
10 mM dNTPs 1.0 9.0
Phusion pol. 0.5 4.5
5x HF buffer 5.0 45.0
dH2O 41.3 371.7
  50.0

Program: Phusion (block A) - edited to include 65°C - 55°C annealing gradient

  • 98°C, 3 min
  • 30x[98°C, 10 sec; 68°C - 57°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞


Conclusions

  • tba




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