User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/21: 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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* Note: unsure why concentrations so low. Proceed with ligations anyway, see what happens.
* Note: unsure why concentrations so low. Proceed with ligations anyway, see what happens.


* Ligations
* 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 -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1   || 2    ||
| &nbsp;            || Rxn 1-4  || Neg
|-
|-
| Insert DNA        || ### || ---  ||
| Insert DNA        || 3.0 || ---   
|-
|-
| Vector DNA         || ### || ### ||
| Vector DNA (25 ng) || 3.5 || 3.5  
|-
|-
| 2x lgn buf (Roche) || ### || ### ||
| 2x lgn buf (Roche) || 7.5 || 7.5  
|-
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0   
|-
|-
| dH<sub>2</sub>O    || ### || ### ||
| dH<sub>2</sub>O    || --- || 3.0  
|-
|-
| &nbsp;            || # μL || # μL ||
| &nbsp;            || 15 μL || 15 μL  
|}
|}
* Transform 50 uL DH5α-turbo
* Plate on 100 μg/mL amp
RESULTS (4/22/15)
# AubR/MRV: 11 colonies
# BjaR/MRV: 11 colonies
# BraR/MRV: 6 colonies
# RpaR/MRV: 7 colonies
# MRV (neg): 3 colonies
* Success! Pick 2 colonies from plates 1-4 for streak plate and liquid cultures (5 mL each)




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| bgcolor=#cfcfcf | Mix(x13)
| bgcolor=#cfcfcf | Mix(x13)
| rowspan=7 | Expected<br>pX330 = ~8335, 195<br>gRNA+pX330 = 8530
| rowspan=7 | Expected<br>pX330 = ~8335, 195<br>gRNA+pX330 = 8530
| rowspan=7 | [[Image:somegel.jpg|250px|Hover label]]
| rowspan=7 | [[Image:KAH042115_gel1.jpg|250px|Hover label]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
|-
| DNA || 3.0 || ---
| DNA || 3.0 || ---
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** Proceed with sequencing
** Proceed with sequencing


# g51-1, f
* Measure conc.'s
# g51-1, r
{| {{table}} cellspacing="3" <!-- [DNA] table -->
# etc.
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. gRNA51-1 || 0.233 || 1.928 || 232.82
|-
| 2. gRNA51-2 || 0.112 || 1.901 || 112.471
|-
| 3. gRNA52-1 || 0.203 || 1.901 || 203.39
|-
| 4. gRNA52-2 || 0.237 || 1.923 || 236.79
|-
| 5. gRNA53-1 || 0.212 || 1.903 || 212.34
|-
| 6. gRNA55-1 || 0.233 || 1.906 || 233.19
|-
| 7. gRNA55-2 || 0.242 || 1.896 || 242.43
|-
| 8. gRNA55-1 || 0.217 || 1.895 || 217.49
|-
| 9. gRNA55-2 || 0.264 || 1.896 || 264.27
|}
 
 
Sequencing list:
# g51-1, f (P139) - Confirmed
# g51-1, r (P140) - low Phred 20 score
# g51-2, f - Confirmed
# g51-2, r - low Phred 20 score
# g52-1, f - Confirmed
# g52-1, r - low Phred 20 score
# g52-2, f - Confirmed
# g52-2, r - low Phred 20 score
# g53-1, f - Confirmed
# g53-1, r - low Phred 20 score
# g55-1, f - Confirmed
# g55-1, r - low Phred 20 score
# g55-2, f  - Confirmed
# g55-2, r - low Phred 20 score
# g56-1, f - Confirmed
# g56-1, r - low Phred 20 score
# <font color="red">g56-2, f - questionable sequence across gRNA, discard
# g56-2, r - low Phred 20 score</font>
 
 
* Cultures for new minipreps
** 6 & 7 were accidentally mixed - discard these
** Set up 5 mL cultures for gRNA53-2 and gRNA54




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'''LCR Development'''
'''LCR Development'''
* Final round of Phusion PCR reactions
* Final round of Phusion PCR reactions
Phusion PCR:
# MV10 + pol
# MV10 + pol
# MV10 no pol (neg ctrl to see how much of band is template)
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1,2
| bgcolor=#cfcfcf | Rxn3
| rowspan="7" | <u>Expected:</u><br>1,2. MV10 = 5191<br>3. No product
| rowspan="7" | [[Image:KAH042215_gel1.jpg|250px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| DNA(clean linear) || 1.0 μL || 1.0 μL
|-
| 10 μM F primer || 1.0 || 1.0
|-
| 10 μM R primer || 1.0 || 1.0
|-
| 10 mM dNTPs || 1.0 || 1.0
|-
| Phusion Pol. || 0.5 || ---
|-
| 5X HF buffer || 10.0 || 10.0
|-
| dH<sub>2</sub>O || 35.5 || 36.0
|-
| &nbsp; || 50.0 || 50.0
|}
Program: Phusion (block A)
* 98°C, 3 min
* 35x[98°C, 10 sec; 65°C, 30 sec; 72°C, 2.5 min]
* 72°C, 10 min
* 4°C ∞
Conclusion
* Lane 3 shows product. Must have accidentally added Phusion pol.
* Desired band not any brighter than reaction done at 60°C (see [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/04/17 04/17/15]).
* Use reactions from 4/17/15 instead of these for LCR.
Phusion PCR:
# Gal4DB-mCh - plasmid
# "
# ATF2 - plasmid template
# "
# ATF2 - 1:10 iPSC cDNA template
# "
{| {{table}} border="1" cellspacing="3" <!-- Phusion PCR table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Rxn1-4
| bgcolor=#cfcfcf | Rxn5,6
| rowspan="7" | <u>Expected:</u><br>5,6. Gal4DB-mCh = 1170<br>7-10. ATF2 = 906
| rowspan="7" | [[Image:KAH042215_gel1.jpg|250px|Hover name]]<br>5 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| DNA(clean linear) || 0.5 μL || 2.0 μL
|-
| 10 μM F primer || 1.0 || 1.0
|-
| 10 μM R primer || 1.0 || 1.0
|-
| 10 mM dNTPs || 1.0 || 1.0
|-
| Phusion Pol. || 0.5 || 0.5
|-
| 5X HF buffer || 10.0 || 10.0
|-
| dH<sub>2</sub>O || 36.0 || 34.5
|-
| &nbsp; || 50.0 || 50.0
|}
Program: Phusion (block B)
* 98°C, 3 min
* 35x[98°C, 10 sec; 55°C, 30 sec; 72°C, 60 sec]
* 72°C, 10 min
* 4°C ∞
Conclusion
* Gal4DB-mCh - much better than previous reactions. Purify these (combined) and use for LCR
* ATF2 - rxns w/ plasmid template look awesome! cDNA templates give a spurious band. Use plasmid PCR's (combined, purified) for LCR.




Latest revision as of 00:55, 27 September 2017

Karmella's BioBrick Cloning Main project page
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04/21/15

  • Ryan - regulator assemblies
  • Rene - check gRNA assemblies; digests & sequencing
  • LCR Development - Phusion PCR


Ryan - Receiver plasmids, assembly

  • Stage 1 - insert Regulator ORFS
    • DONE - Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
    • Insert ORF(E/X) into Vector (E/X). see Vector plasmid map
  • Stage 2 - insert Promoters
    • Cut & dephos promoters with E/X
    • Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


  • Clean up PCR products
    • Zymo clean and concentrator
    • Elute w/ 25 μL dH2O


  • Assemblies
  1. AubR/MRV: AubR-PCR(E/X)/851 + MRV(E/X)/3201
  2. BjaR/MRV: BjaR-PCR(E/X)/776 + MRV(E/X)/3201
  3. BraR/MRV: BraR-PCR(E/X)/776 + MRV(E/X)/3201
  4. RpaR/MRV: RpaR-PCR(E/X)/779 + MRV(E/X)/3201


  • Digests
    • E/X
  1. AubR, 851 bp
  2. BjaR, 776 bp
  3. BraR, 776 bp
  4. RpaR, 779 bp
  5. Modular Receiver Vector (MRV)
Reagent Rxn 1-4 Rxn 5
DNA 10.0 5.0
10X buffer 3.0 3.0
EcoRI 1.0 1.0
XbaI 1.0 1.0
dH2O 15.0 20.0
  30.0 30.0


  • Column clean-up
    • Zymo clean and concentrator


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. AubR-PCR (E/X) -0.005 5.875 -5.001
2. BjaR-PCR (E/X) -0.005 9 -4.788
3. BraR-PCR (E/X) -0.005 7.143 -5.291
4. RpaR-PCR (E/X) -0.003 29 -3.052
5. MRV (E/X) 0.007 1.388 7.11
  • Note: unsure why concentrations so low. Proceed with ligations anyway, see what happens.


  • Ligations
  Rxn 1-4 Neg
Insert DNA 3.0 ---
Vector DNA (25 ng) 3.5 3.5
2x lgn buf (Roche) 7.5 7.5
T4 ligase (NEB) 1.0 1.0
dH2O --- 3.0
  15 μL 15 μL
  • Transform 50 uL DH5α-turbo
  • Plate on 100 μg/mL amp

RESULTS (4/22/15)

  1. AubR/MRV: 11 colonies
  2. BjaR/MRV: 11 colonies
  3. BraR/MRV: 6 colonies
  4. RpaR/MRV: 7 colonies
  5. MRV (neg): 3 colonies
  • Success! Pick 2 colonies from plates 1-4 for streak plate and liquid cultures (5 mL each)


Rene - check gRNA assemblies

  • Minipreps from 4/20/15
    • Note: ran out of time last week to grow up cultures. Stored at 4C until this week, grew for ~5 hours and then miniprepped w/ Sigma GenElute kit (elution vol. = 75 μL)
  • Check w/ BpiI/XbaI
  1. gRNA51, 1
  2. gRNA51, 2
  3. gRNA52, 1
  4. gRNA52, 2
  5. gRNA53, 1
  6. gRNA53, 2 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!)
  7. gRNA54 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!)
  8. gRNA55, 1
  9. gRNA55, 2
  10. gRNA56, 1
  11. gRNA56, 2
  12. pX330 (ctrl)


Reagent Rxn 1-11 Mix(x13) Expected
pX330 = ~8335, 195
gRNA+pX330 = 8530 		Hover label
15 μL/lane; 1% agarose; Ladder
DNA 3.0 ---
10X buffer 1.5 19.5
BpiI 0.5 6.5
XbaI 0.5 6.5
dH2O 9.5 123.5
  15.0


  • Conclusion
    • Gel inconclusive, expected sizes not seen for pX330 control
    • Proceed with sequencing
  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. gRNA51-1 0.233 1.928 232.82
2. gRNA51-2 0.112 1.901 112.471
3. gRNA52-1 0.203 1.901 203.39
4. gRNA52-2 0.237 1.923 236.79
5. gRNA53-1 0.212 1.903 212.34
6. gRNA55-1 0.233 1.906 233.19
7. gRNA55-2 0.242 1.896 242.43
8. gRNA55-1 0.217 1.895 217.49
9. gRNA55-2 0.264 1.896 264.27


Sequencing list:

  1. g51-1, f (P139) - Confirmed
  2. g51-1, r (P140) - low Phred 20 score
  3. g51-2, f - Confirmed
  4. g51-2, r - low Phred 20 score
  5. g52-1, f - Confirmed
  6. g52-1, r - low Phred 20 score
  7. g52-2, f - Confirmed
  8. g52-2, r - low Phred 20 score
  9. g53-1, f - Confirmed
  10. g53-1, r - low Phred 20 score
  11. g55-1, f - Confirmed
  12. g55-1, r - low Phred 20 score
  13. g55-2, f - Confirmed
  14. g55-2, r - low Phred 20 score
  15. g56-1, f - Confirmed
  16. g56-1, r - low Phred 20 score
  17. g56-2, f - questionable sequence across gRNA, discard
  18. g56-2, r - low Phred 20 score


  • Cultures for new minipreps
    • 6 & 7 were accidentally mixed - discard these
    • Set up 5 mL cultures for gRNA53-2 and gRNA54


LCR Development

  • Final round of Phusion PCR reactions

Phusion PCR:

  1. MV10 + pol
  2. MV10 + pol
  3. MV10 no pol (neg ctrl to see how much of band is template)
Reagent Rxn1,2 Rxn3 Expected:
1,2. MV10 = 5191
3. No product 		Hover name
5 μL/lane; 1% agarose; Ladder
DNA(clean linear) 1.0 μL 1.0 μL
10 μM F primer 1.0 1.0
10 μM R primer 1.0 1.0
10 mM dNTPs 1.0 1.0
Phusion Pol. 0.5 ---
5X HF buffer 10.0 10.0
dH2O 35.5 36.0
  50.0 50.0

Program: Phusion (block A)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 65°C, 30 sec; 72°C, 2.5 min]
  • 72°C, 10 min
  • 4°C ∞


Conclusion

  • Lane 3 shows product. Must have accidentally added Phusion pol.
  • Desired band not any brighter than reaction done at 60°C (see 04/17/15).
  • Use reactions from 4/17/15 instead of these for LCR.


Phusion PCR:

  1. Gal4DB-mCh - plasmid
  2. "
  3. ATF2 - plasmid template
  4. "
  5. ATF2 - 1:10 iPSC cDNA template
  6. "
Reagent Rxn1-4 Rxn5,6 Expected:
5,6. Gal4DB-mCh = 1170
7-10. ATF2 = 906 		Hover name
5 μL/lane; 1% agarose; Ladder
DNA(clean linear) 0.5 μL 2.0 μL
10 μM F primer 1.0 1.0
10 μM R primer 1.0 1.0
10 mM dNTPs 1.0 1.0
Phusion Pol. 0.5 0.5
5X HF buffer 10.0 10.0
dH2O 36.0 34.5
  50.0 50.0

Program: Phusion (block B)

  • 98°C, 3 min
  • 35x[98°C, 10 sec; 55°C, 30 sec; 72°C, 60 sec]
  • 72°C, 10 min
  • 4°C ∞


Conclusion

  • Gal4DB-mCh - much better than previous reactions. Purify these (combined) and use for LCR
  • ATF2 - rxns w/ plasmid template look awesome! cDNA templates give a spurious band. Use plasmid PCR's (combined, purified) for LCR.




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