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		+	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.
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Revision as of 17:03, 6 June 2011

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 4 Thursday 4.1 Reporters 4.1.1 Miniprep 4.1.2 PCR 4.1.3 Nanodrop

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/ml)	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.

Back to Notebook