Team:BYU Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry
From 2013.igem.org
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::: - 6uL of each primer (BI309 and BI310) | ::: - 6uL of each primer (BI309 and BI310) | ||
:: * Mix well | :: * Mix well | ||
- | :: * To six new PCR tubes, labeled S4, S10, S21, L8, | + | :: * To six new PCR tubes, labeled S4, S10, S21, L8, WT, and control respectively, add 2uL of template DNA from the boiled samples. |
:: * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix). | :: * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix). | ||
:: * Add 48uL of the master mix into each PCR tube. | :: * Add 48uL of the master mix into each PCR tube. | ||
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:: * a regular gel was used to verify PCR product | :: * a regular gel was used to verify PCR product | ||
:: * 3uL of product and 3uL of loading dye was used per well in the gel. | :: * 3uL of product and 3uL of loading dye was used per well in the gel. | ||
+ | |||
+ | 3) Restriction digest (10.21) | ||
+ | : * Followed procedure for PCR cleanup of WT, S4, S10, S21, and L8. | ||
+ | : * 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 added part of the vector digestion tube to each of the individual tubes. | ||
+ | : * The five individual tubes were placed in the 37C incubator overnight. | ||
+ | |||
+ | 4) Low-melt Gel for Purification of Sticky-ended Insert/Vector (10.22) | ||
+ | : * Ran a low-melt gel for each of the individual tubes. | ||
+ | : * Cut out each bright band and placed them in an eppendorf tube. | ||
+ | : * Left them in the freezer overnight. | ||
+ | |||
+ | 5) Ligation (10.23) | ||
+ | : * Centrifuged the eppendorf tubes from step 4 to separate the | ||
+ | |||
+ | 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''' | '''V) Results''' |
Revision as of 02:42, 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)
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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