Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/30: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Ligation/Amplification of DBN002 & DBN003</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Ligation/Amplification of DBN002 & DBN003</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}}}}]]}} | ||
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<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
Going to try Type IIS Digestion/Ligation of DBN002 & DBN003, followed by PCR amplification. | Going to try Type IIS Digestion/Ligation of DBN002 & DBN003, followed by PCR amplification. Also going to try conventional assembly using the [http://openwetware.org/wiki/Haynes:Assembly101 BioBricks method ]. | ||
==Type IIS Digestion/Ligation== | ==Type IIS Digestion/Ligation== | ||
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Incubate the ligation reaction in a thermocycler: | Incubate the ligation reaction in a thermocycler (program name DBN IIS): | ||
# 37°C 5 min | # 37°C 5 min | ||
# 23°C 5 min | # 23°C 5 min | ||
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# 4°C hold until ready to proceed | # 4°C hold until ready to proceed | ||
<b>--Clean-up of IIS Ligation--</b> | |||
Use the Qiagen kit. If possible, final volume elution of 30 µL. | Use the Qiagen kit. If possible, final volume elution of 30 µL. | ||
<b>--PCR--</b> | |||
{| {{table}} | {| {{table}} | ||
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| soak||4||indef. | | soak||4||indef. | ||
|} | |} | ||
==BioBricks Cloning== | |||
Follow the method described [http://openwetware.org/wiki/Haynes:Assembly101 here]. | |||
Parts to be used are KAH57 (AmCyan, AmCyan, NLS, Stop) and KAH104 (HPK, Kozak). Both are in v0120 vector. For this cloning, KAH57 will be the vector and KAH104 the insert, placed in front of the KAH57 part. Enzymes used will be E/X for KAH57 and E/S for KAH104. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (KAH57)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (KAH104)''' | |||
|- | |||
| DNA||25||15 | |||
|- | |||
| 10x Green FD Buffer||3||3 | |||
|- | |||
| EcoRI||1||1 | |||
|- | |||
| SpeI||0||1 | |||
|- | |||
| XbaI||1||0 | |||
|- | |||
| Water||0||10 | |||
|- | |||
| Total||30||30 | |||
|} | |||
During gel extraction, remove the vector from KAH57 (should be only large piece in digest) & the insert from KAH104 (540bp). | |||
Gel image: | |||
[[Image:2015-04-30_KAH57_%26_KAH104_digests_annotated.png]] | |||
Cut out vector (0.134g) and insert (0.140g). Ran a gel extraction using Sigma GenElute Gel Extraction Kit, then measured concentration on the spectrophotometer. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/µL)''' | |||
| align="center" style="background:#f0f0f0;"|'''St Dev (ng/µL)''' | |||
| align="center" style="background:#f0f0f0;"|'''260/280''' | |||
|- | |||
| KAH57||5.128||1.458054183||2.2275 | |||
|- | |||
| KAH104||7.9305||0.907218||2.451 | |||
|} | |||
Purity as measured by A260/A280 is poor, and concentration is low. Proceeding with ligation step. | |||
50ng vector * 1/5.1 ng/µL = 10 µL purified KAH57 | |||
ng insert = 540bp/4691bp * 2 * 50ng = 11.5 ng, * 1/7.9 ng/µL = 1.5 µL purified KAH104 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (µL)''' | |||
|- | |||
| vector||10 | |||
|- | |||
| insert||1.5 | |||
|- | |||
| Roche Buffer 2x||10 | |||
|- | |||
| NEB T4 ligase||2 | |||
|- | |||
| Total||23.5 | |||
|} | |||
Incubate for 10 minutes at room temperature. | |||
==Transformation== | |||
Transform DH5α-Turbo with: | |||
* ✓ KAH57_v0120 | |||
* ✓ KAH104_v0120 | |||
* ✓ pMax-GFP (brochure says it needs a Kanamycin plate, but René says she used Amp, so try that first) | |||
* ✓ ligation product of KAH57 & KAH104 | |||
* ✓ negative control (water) | |||
* ✓ negative control (digested KAH57 only) | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
|} | |} | ||
__NOTOC__ | __NOTOC__ |
Latest revision as of 00:56, 27 September 2017
Ligation/Amplification of DBN002 & DBN003 | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
OverviewGoing to try Type IIS Digestion/Ligation of DBN002 & DBN003, followed by PCR amplification. Also going to try conventional assembly using the BioBricks method . Type IIS Digestion/Ligation
Incubate the ligation reaction in a thermocycler (program name DBN IIS):
--Clean-up of IIS Ligation-- Use the Qiagen kit. If possible, final volume elution of 30 µL. --PCR--
BioBricks CloningFollow the method described here. Parts to be used are KAH57 (AmCyan, AmCyan, NLS, Stop) and KAH104 (HPK, Kozak). Both are in v0120 vector. For this cloning, KAH57 will be the vector and KAH104 the insert, placed in front of the KAH57 part. Enzymes used will be E/X for KAH57 and E/S for KAH104.
During gel extraction, remove the vector from KAH57 (should be only large piece in digest) & the insert from KAH104 (540bp). Gel image: Cut out vector (0.134g) and insert (0.140g). Ran a gel extraction using Sigma GenElute Gel Extraction Kit, then measured concentration on the spectrophotometer.
Purity as measured by A260/A280 is poor, and concentration is low. Proceeding with ligation step. 50ng vector * 1/5.1 ng/µL = 10 µL purified KAH57 ng insert = 540bp/4691bp * 2 * 50ng = 11.5 ng, * 1/7.9 ng/µL = 1.5 µL purified KAH104
Incubate for 10 minutes at room temperature. TransformationTransform DH5α-Turbo with:
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