Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/30: Difference between revisions
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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. | ||
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==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)''' | |||
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| 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). | |||
==Transformation== | |||
Transform DH5α-Turbo with: | |||
* KAH57 | |||
* KAH104 | |||
* pMax-GFP | |||
* ligation product of KAH57 & KAH104 | |||
* ligation product of DBN002 & DBN003 | |||
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__NOTOC__ | __NOTOC__ |
Revision as of 15:16, 30 April 2015
Ligation/Amplification of DBN002 & DBN003 | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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). TransformationTransform DH5α-Turbo with:
|