User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/21: Difference between revisions
From OpenWetWare
(Autocreate 2015/04/21 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning) |
(fix raw html notebook nav) |
||
(24 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|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"| | |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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==04/21/15== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* | * 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 [https://benchling.com/s/VFNzuauP/edit 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 dH<sub>2</sub>O | |||
* Assemblies | |||
# AubR/MRV: AubR-PCR(E/X)/851 + MRV(E/X)/3201 | |||
# BjaR/MRV: BjaR-PCR(E/X)/776 + MRV(E/X)/3201 | |||
# BraR/MRV: BraR-PCR(E/X)/776 + MRV(E/X)/3201 | |||
# RpaR/MRV: RpaR-PCR(E/X)/779 + MRV(E/X)/3201 | |||
* Digests<br> | |||
** E/X | |||
# AubR, 851 bp | |||
# BjaR, 776 bp | |||
# BraR, 776 bp | |||
# RpaR, 779 bp | |||
# Modular Receiver Vector (MRV) | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | ||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | Rxn 1-4 | ||
| bgcolor=#cfcfcf | Rxn 5 | |||
| | |||
|- | |- | ||
| DNA | | DNA || 10.0 || 5.0 | ||
|- | |- | ||
| 10X buffer || | | 10X buffer || 3.0 || 3.0 | ||
|- | |- | ||
| EcoRI || 1.0 | | EcoRI || 1.0 || 1.0 | ||
|- | |- | ||
| | | XbaI || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 15.0 || 20.0 | ||
|- | |- | ||
| || | | || 30.0 || 30.0 | ||
|} | |} | ||
* Column clean-up | |||
** Zymo clean and concentrator | |||
* Measure conc.'s | |||
{| {{table}} cellspacing="3" <!-- [DNA] table --> | |||
|- bgcolor=#cfcfcf | |||
| 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 | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | |||
| || 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 | |||
|- | |||
| dH<sub>2</sub>O || --- || 3.0 | |||
|- | |||
| || 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) | |||
---- | ---- | ||
''' | '''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 | |||
# gRNA51, 1 | |||
# gRNA51, 2 | |||
# gRNA52, 1 | |||
# gRNA52, 2 | |||
# gRNA53, 1 | |||
# gRNA53, 2 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!) | |||
# gRNA54 (Note: Accidentally mixed 6 w/ 7 during pellet resuspension!) | |||
# gRNA55, 1 | |||
# gRNA55, 2 | |||
# gRNA56, 1 | |||
# gRNA56, 2 | |||
# pX330 (ctrl) | |||
{| {{table}} cellspacing="3" <!-- Digest rxn. table --> | |||
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table --> | |||
|- valign="top" | |- valign="top" | ||
| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | | | bgcolor=#cfcfcf | Rxn 1-11 | ||
| rowspan= | | bgcolor=#cfcfcf | Mix(x13) | ||
| rowspan=7 | Expected<br>pX330 = ~8335, 195<br>gRNA+pX330 = 8530 | |||
| 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 | | DNA || 3.0 || --- | ||
|- | |- | ||
| | | 10X buffer || 1.5 || 19.5 | ||
|- | |- | ||
| | | BpiI || 0.5 || 6.5 | ||
|- | |- | ||
| | | XbaI || 0.5 || 6.5 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 9.5 || 123.5 | ||
|- | |- | ||
| || | | || 15.0 || | ||
|} | |} | ||
* Conclusion | |||
** Gel inconclusive, expected sizes not seen for pX330 control | |||
** Proceed with sequencing | |||
* Measure conc.'s | * Measure conc.'s | ||
Line 70: | Line 172: | ||
| Sample || OD260 || 260/280 || ng/μL | | Sample || OD260 || 260/280 || ng/μL | ||
|- | |- | ||
| 1. | | 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: | ||
{| {{table}} cellspacing="3" <!-- | # g51-1, f (P139) - Confirmed | ||
|- bgcolor=#cfcfcf | # 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 | |||
---- | |||
'''LCR Development''' | |||
* 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] | |||
|- | |- | ||
| 1. | | 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 | | dH<sub>2</sub>O || 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 [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 | |||
|- | |||
| || 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 Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
04/21/15
Ryan - Receiver plasmids, assembly
RESULTS (4/22/15)
Rene - check gRNA assemblies
LCR Development
Phusion PCR:
Program: Phusion (block A)
Program: Phusion (block B)
|