Team:BYU Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry
From 2013.igem.org
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: * For each PCR product (WT, S4, S10, S21, and L8), we set up an individual tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of PCR product, and 2ul of each restriction enzyme. | : * For each PCR product (WT, S4, S10, S21, and L8), we set up an individual tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of PCR product, and 2ul of each restriction enzyme. | ||
: * We also made a vector digestion tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of the vector digestion, and 2ul of each restriction enzyme. | : * We also made a vector digestion tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of the vector digestion, and 2ul of each restriction enzyme. | ||
- | + | : * The six tubes were placed in the 37C incubator overnight. | |
- | : * The | + | |
4) Low-melt Gel for Purification of Sticky-ended Insert/Vector (10.22) | 4) Low-melt Gel for Purification of Sticky-ended Insert/Vector (10.22) | ||
- | : * Ran a low-melt gel for each of the | + | : * Ran a low-melt gel for each of the tubes in step 3. |
: * Cut out each bright band and placed them in an eppendorf tube. | : * Cut out each bright band and placed them in an eppendorf tube. | ||
: * Left them in the freezer overnight. | : * Left them in the freezer overnight. | ||
5) Ligation (10.23) | 5) Ligation (10.23) | ||
- | : * Centrifuged the eppendorf tubes from step 4 to separate the | + | : * Centrifuged the eppendorf tubes from step 4 to separate the vector or insert from the gel. |
+ | : * Created 5 ligation reactions for each phage (WT, S4, S10, S21, and L8) with 6.5ul water, 1.5ul 10X ligase buffer, 1ul T4 DNA ligase, 3ul vector, and 3ul insert. | ||
+ | |||
+ | 6) Transformation ( | ||
Monday: Restriction digest | Monday: Restriction digest |
Revision as of 02:46, 29 October 2013
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10.18 Cloning T7 Capsid Protein into iGEM Registry
I) Purpose
II) Expected Outcome
III) Reagents Used
IV) Procedure 1) Amplification and purification of insert (10.18)
2) Repeat of the amplification and purification of insert (10.20)
3) Restriction digest (10.21)
4) Low-melt Gel for Purification of Sticky-ended Insert/Vector (10.22)
5) Ligation (10.23)
6) Transformation ( Monday: Restriction digest Tuesday: Low-melt gel for purification of sticky-ended insert/vector (Jade) Wednesday: Ligation and transformation Thursday: Redid plating for transformation and started overnights of each colony and ran gel Friday: Cleaned up the plasmid and sent it in V) Results 1) Amplification and purification of insert
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