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From 2011.igem.org

Contents 1 Monday 1.1 Reporters 1.1.1 PCR 1.1.2 Restriction Digest 1.1.3 Ligation 1.1.4 Transformation/Plating 2 Tuesday 2.1 Reporters 2.1.1 Colony PCR 2.1.2 Agarose Gel Electrophoresis 2.1.3 Restriction Digest 2.1.4 Ligation 2.1.5 Transformation/Plating 3 Wednesday 3.1 Reporters 3.1.1 Colony PCR 3.1.2 Agarose Gel Electrophoresis 4 Thursday 4.1 Reporters 4.1.1 Miniprep 4.1.2 PCR 4.1.3 Nanodrop 4.1.4 Restriction Digest 4.1.5 Ligation 4.1.6 Transformation/Plating 5 Friday 5.1 Reporters 5.1.1 Colony PCR 5.1.2 Agarose Gel Electrohporesis 5.1.3 PCR Assembly 5.1.4 Culture 6 Saturday 6.1 Reporters 6.1.1 PCR Assembly

Monday

Reporters

PCR

     J33204 miniprep from 5/26/11

Restriction Digest

• Insert with XbaI and PstI

     J33204

• Vector with SpeI and PstI

     R0010

Ligation

     J33204+R0010 digests

Transformation/Plating

     The above ligation was transformed into Escherichia coli cells and plated on Amp resistant plates.

Tuesday

Reporters

Colony PCR

     Two colonies from plates from 5/30/11

Agarose Gel Electrophoresis

1. J33240 PCR products
2. Colony PCR products from above

• Results: The gel showed that the insert (J33240) PCR worked, but the assembly did not work. We came to this conclusion because the constructs on the gel were less than 500 base pairs and our desired construct should have been about 1100 base pairs. Thus, we needed to do assembly for a third time. We started this third assembly with the R0100 miniprep from 5/26/11 and the J33204 PCR products.

Restriction Digest

• Insert with XbaI and PstI

     J33204

• Vector with SpeI and PstI

     R0010

Ligation

     J33204+R0010 digests

Transformation/Plating

     The above ligation was transformed into Escherichia coli cells and plated onto Amp resistant plates.

Wednesday

Reporters

Colony PCR

     Colonies from plates from 5/31/11

Agarose Gel Electrophoresis

     Colony PCR products from above

• The gel showed that the assembly did not work. The gel had the same results of the gel from last week and the one from 5/31/11. We determined that we need to use a more specific ratio of insert DNA to vector DNA. We will determine this ratio in our next assembly. The next assembly began with transformation of J33204 and R0010 from the registry into Escherichia coli cells.

Thursday

Reporters

Miniprep

1. J33204
2. R0010

PCR

     J33204

Nanodrop

     We needed to determine the concentration of DNA in our miniprep products, so we used a nanodrop. Our results are shown in the following table.

Registry Part	Concentration (ng/μl)	260/280
J33240	22.9	1.93
R0010	137.3	1.90

We used this information to calculate the concentration (in picomoles/microliter) of our insert and vector DNA. The calculation we used was:

(concentration of DNA)/[(0.66)*(size of part in base pairs)]

We wanted 0.25 pM of vector and 0.50 pM of insert, so we calculated how much volume of the insert and vector digests we needed to add for our ligation. These volumes as well as the molar concentrations of our minipreps are summarized in the below table.

Registry Part	Molar Concentration (pM/μl)	Volume for Ligation (μl)
J33204	0.0119	21.0
R0010	0.223	2.24

Restriction Digest

• Insert with XbaI and PstI

     J33204

• Note: the following changes were made from the digest protocol:

dH20:	39.26 μl
J33204:	2.24 μl

• Vector with SpeI and PstI

     R0010

• Note: the following changes were made from the digest protocol:

dH20:	21.0 μl
R0010:	21.0 μl

Ligation

To get a 4:1 ratio of insert DNA to vector DNA, the following changes were made to the ligation protocol:

Insert (J33240):	2 μl
Vector (R0010):	4 μl

To get a 6:1 ratio of insert DNA to vector DNA, the following changes were made to the ligation protocol:

Insert (J33204):	1.5 μl
Vector (R0010):	4.5 μl

Transformation/Plating

     The above ligation was transformed into Escherichia coli cells and plated onto Amp resistant plates.

Friday

Reporters

Colony PCR

1. 4:1 ratio colonies
2. 6:1 ratio colonies

• Note: the 4:1 ratio colonies grew a lot more than the 6:1 colonies

Agarose Gel Electrohporesis

1. 4:1 colony PCR products
2. 6:1 colony PCR products

Results: One of the 6:1 ratio constructs contained the correct amount of base pairs (about 1100). The other constructs did not have more than 500 base pairs. The 6:1 ratio construct will be sequenced to determine its legitimacy.

PCR Assembly

     Now that our primers, which were designed last week, arrived, we started constructing the linkers and cleavage sites via PCR. The registry names and our colloquial names (names we refer to these parts as throughout this notebook) are summarized in the following table. We followed the Assembly via PCR protocol for these assemblies.

Registry Name	Colloquial Name
K243004	small linker
K105012	long linker
K316007	Imperial linker
N/A	tev cleavage site
N/A	cI cleavage site

• Note: When purifying the PCR products, DNA wash buffer without ethanol was used, making all of the PCR products unusable.

Culture

     The 6:1 ratio colony that seemed to work based on the gel electrophoresis was grown overnight.

Saturday

Reporters

PCR Assembly

      The PCR assembly and parts from 6/3/11 were repeated. The correct purification occurred. A gel electrophoresis analysis showed that the PCR assemblies worked.

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