Team:BYU Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry
From 2013.igem.org
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: * 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. | : * 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 ( | + | 6) Transformation (10.23) |
+ | : * Added 2ul of ligation mix to 25ul of competent cells. Vortexed the tubes briefly and placed them on ice for 10 minutes. | ||
+ | : * Heat shocked at 42C for 1 minute and then placed tubes back on ice for 10 minutes. | ||
+ | : * Added 0.5mL of plain LB to the reactions and incubated at 37C for 1 hour. | ||
+ | : * Plated 100ul of cells on CAM plates and incubated at 37C overnight. | ||
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+ | 7) Colony PCR and Overnights for Vector Purification (10.24) | ||
+ | : * Our transformations only had WTa, WTb, S4, S10a, S10b, S10c, S21, L8a, and L8b (a,b,c designates multiple colonies from the same transformation. | ||
+ | : * For each colony, we picked it with a pipet tip, dipped it in 50ul of water in a PCR tube, and placed the tip in a test tube of 5mL of LB (These tubes were placed in the 37C incubator as overnights). | ||
+ | : * Boil each PCR tube for 5 minutes. | ||
+ | : * Set up a PCR reaction for each PCR tube by adding 2.5ul Standard 10X reaction buffer, 0.5ul 10mM dNTP's, 0.5ul of each primer, 0.5ul Taq DNA polymerase, and 2ul of boiled colony sample. | ||
+ | : * Run PCR for 25 cycles. | ||
+ | : * Run a gel with 5ul of each PCR product to check for proper size. | ||
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+ | 8) Plasmid Cleanup (10.25) | ||
+ | : * Followed procedure for plasmid cleanup for WT, S4, S10, and L8. | ||
+ | : * Mailed 10ul of each plasmid to iGEM! | ||
Monday: Restriction digest | Monday: Restriction digest |
Revision as of 03:15, 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 (10.23)
7) Colony PCR and Overnights for Vector Purification (10.24)
8) Plasmid Cleanup (10.25)
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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