User:Karmella Haynes/Notebook/BioBrick cloning/2013/02/23: Difference between revisions

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----
----
'''Type IIS Assembly: H2B-LOV'''
'''Type IIS Assembly, PCR: LOV-H2B+his'''
# BioBrick name: LOV/(gg)/size + 3' part/(c/d)/size
# H2B = 378 bp, ggH2B f/r (Template: H2B-GFP from Addgene); note: reverse primer adds a 6-his tag (18 bp)
# LOV = 429 bp, ggLOV f/r
# pSB1A3, gg0001/0002 v2


 
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
 
* PCR (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 | 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] -->
| rowspan="7" | <u>Expected:</u><br>1. pSB1A3 = ~2000<br>2. hPCD = 186<br>3. BL01 = 2520
| rowspan="7" | [[Image:KAH011013_gel1.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
| DNA(plasmid) || 0.2 μL
|-
|-
| 10x buffer || 3.0
| primer 1 || 1.0
|-
|-
| enzyme 1 || 1.0
| primer 2 || 1.0
|-
|-
| enzyme 2 || 1.0
| 2x GoTaq || 25.0
|-
|-
| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || 22.8
|-
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
| &nbsp; || 50.0 μL
|}
|}


PCR
* 95°C, 3 min.
* [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
* 72°C, 3 min.
* 4°C, ∞


* Measure conc.'s
 
{| {{table}} cellspacing="3" <!-- [DNA] table -->
Purification
|- bgcolor=#cfcfcf  
* Clean with Zymo DNA c&c; elute w/ 20 μL dH<sub>2</sub>O
| Sample || OD260 || 260/280 || ng/μL
* Measure [DNA]
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Sample
| bgcolor=#cfcfcf | OD 260
| bgcolor=#cfcfcf | 260/280  
| bgcolor=#cfcfcf | ng/μL  
|-
| 1. pSB1A3 || 0.126 || 1.888 || 77.357
|-
|-
| 1. Digested part (a/b) || --- || --- || ---
| 2. hPCD || 0.069 || 2.192 || 17.025
|-
|-
| 2. Digested part (c/d) || --- || --- || ---
| 3. BL01 || 0.121 || 1.898 || 66.878
|}
|}




* Dephosphorylation (Roche)
Dilutions: to final concentration of 20 fmol/μL
{| {{table}} cellspacing="3" <!-- Dephos table -->
{| {{table}} border="1" cellspacing="3" <!-- Dilution table -->
|-
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | DNA
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | length (bp)
|-
| bgcolor=#cfcfcf | ng/μL
| DNA (clean digest) || up to 17 μL (500 ng)
| bgcolor=#cfcfcf | Vol.
|-
| bgcolor=#cfcfcf | + dH<sub>2</sub>O
| 10x buffer d.p. || 2.0
|-
|-
| phosphatase || 1.0
| pSB1A3 || 2000 || 77.4 || 6.7 || 13.3
|-
|-
| dH<sub>2</sub>O || ---
| hPCD || 186 || 17.0 || 2.8 || 17.2
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| BL01 || 2520 || 66.9 || 9.8 || 10.2
|}
|}




* Ligations
Golden Gate Reactions
{| {{table}} cellspacing="3" <!-- Ligations table -->
# hPCD + BL01 + pSB1A3
|- bgcolor=#cfcfcf
# pSB1A3
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
 
{| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
|- valign="top"
| Reagent || 1 || 2
|-
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
| gg2 pSB1A3 || 1.0 || 1.0
|-
|-
| 2. vector(c/d)/ ## ng || &nbsp;
| gg3 hPCD || 1.0 || ---  
|}
 
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
|-
|-
| Insert DNA        || ###  || --- ||
| gg4 BL01 || 1.0 || ---  
|-
|-
| Vector DNA        || ###  || ###  ||
| 10x ligase buffer || 1.0 || 1.0
|-
|-
| 2x lgn buf (Roche) || ###  || ###  ||
| NEB T4 lgase || 0.25 || 0.25
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| BsmBI || 0.5 || 0.5
|-
|-
| dH<sub>2</sub>O   || ###  || ###  ||
| dH<sub>2</sub>O || 5.25 || 7.25
|-
|-
| &nbsp;             || # μL || # μL ||
| &nbsp; || 10.0 || 10.0
|}
|}


----
Thermal cycling
'''Oligo annealing'''
* [45°C, 2 min.; 16°C, 5 min.] x25
# New BB 1
* 60°C, 20 min.
# New BB 2
* 80°C, 20 min.
 
* 4°C, ∞
{| 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 16:26, 23 February 2013

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02/23/13

  • Type IIS Assembly, PCR: LOV-H2B+his


Type IIS Assembly, PCR: LOV-H2B+his

  1. H2B = 378 bp, ggH2B f/r (Template: H2B-GFP from Addgene); note: reverse primer adds a 6-his tag (18 bp)
  2. LOV = 429 bp, ggLOV f/r
  3. pSB1A3, gg0001/0002 v2
Reagent Volume Expected:
1. pSB1A3 = ~2000
2. hPCD = 186
3. BL01 = 2520 		Hover name
10 μL/lane; 1% agarose; Ladder
DNA(plasmid) 0.2 μL
primer 1 1.0
primer 2 1.0
2x GoTaq 25.0
dH2O 22.8
  50.0 μL

PCR

  • 95°C, 3 min.
  • [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 1 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞


Purification

  • Clean with Zymo DNA c&c; elute w/ 20 μL dH2O
  • Measure [DNA]
Sample OD 260 260/280 ng/μL
1. pSB1A3 0.126 1.888 77.357
2. hPCD 0.069 2.192 17.025
3. BL01 0.121 1.898 66.878


Dilutions: to final concentration of 20 fmol/μL

DNA length (bp) ng/μL Vol. + dH2O
pSB1A3 2000 77.4 6.7 13.3
hPCD 186 17.0 2.8 17.2
BL01 2520 66.9 9.8 10.2


Golden Gate Reactions

  1. hPCD + BL01 + pSB1A3
  2. pSB1A3
Reagent 1 2
gg2 pSB1A3 1.0 1.0
gg3 hPCD 1.0 ---
gg4 BL01 1.0 ---
10x ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

Thermal cycling

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



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