From 2009.igem.org

Revision as of 08:12, 30 September 2009

Plan

Aim: Create yeast cells that express LDLR on their cell membrane

Methods:

1. Clone the LDLR gene.
   
2. Create biobrick of LDLR.
   

7/6

Overview:
1.PCR LDLR gene from plasmid containing LDLR cDNA
2.gel-purify DNA from the PCR product
3.insert the DNA to vector (iGEM parts)
4.transform into E. coli and select for ampicillin resistance
5.check for transformation success using colony PCR by LDLR primers

Constructs to be created:
pGal1-Kozak-LDLR-terminator
Obtaining DNA:

Resuspended DNA in the following wells with 10ul water:

plate 1 7D
pGal1(including Kozak sequence)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_J63006 Gal1 promoter]
Transformed 1ul of each of the above into DH5a competent cells:

Transformation

• Mix 1ul of DNA with 100ul of competent cells on ice.
• Leave on ice for 30 minutes.
• Heat shock at 42℃ for 45 seconds.
• Leave on ice for 2 minutes.
• Add 500ul of LB and incubate at 37℃ for 1 hour.
• Plate on LB-ampicillin plates.

7/7

Miniprep of E.coli cells containing LDLR gene with Promega, Wizard Plus SV Miniprep DNA Purification System

Successful

7/19

PCR of LDLR

0.4ul 50uM 5’primer
0.4ul 50uM 3’primer
1.6ul 2.5mM dNTP
2ul 10×Pfu Ultra 2 buffer
0.05ul LDLR plasmid
0.5ul Pfu Ultra
15.05ul MilliQ water
↓
Performed PCR using the following program:
1. 95℃ 2min
2. 95℃ 30sec
3. 55℃ 30sec
4. 72.5℃ 60sec
5. repeat 2-4 29times
6. 25℃ ∞

PCR successful
We cut the region of LDLR out of the gel and purified PCR product using Promega kit.

infusion of LDLR

4ul PCR product (12.35ug/ml)
3ul vector (iGEM parts plate1 D-3, 14.95ug/ml)
2ul 5×infusion reaction buffer
1ul infusion enzyme
↓
37℃ 15min
50℃ 15min
↓
TE buffer up to 20ul
↓
added 10ul of the sample to DH5α (090614) and put on ice for 15 min.
↓
42℃ 45sec
↓
added 500ul LB broth to the tube.
↓
placed it on LB ampicilin plate.

7/20

colony PCR

put a small amount of single colony into each tube with 5ul MilliQ water
↓
95℃ 5min
↓
PCR reaction
1ul 10×buffer
0.8ul 2.5mMdNTP
0.08ul Ex-Taq
0.1ul 5’-primer
0.1ul 3’-primer
2.92ul MilliQ water
↓
added the PCR reaction to each tube.
↓
Performed PCR using the following program:
1. 95℃ 2min
2. 95℃ 30sec
3. 55℃ 30sec
4. 72.5℃ 120sec
5. repeat 2-4 29times
6. 25℃ ∞

PCR unsuccessful・・・.

7/25

gal1 sequencing by BIG DYE

1.8ul 5xB.D.3.1.buffer
0.4ul B.D.3.1.
6.3ul MilliQ water
1ul plasmid(0.15ug/ul)
0.5ul 5'or3'primer(3.2pmol/ul)
↓
PCR　Program
1.96℃ 2min
2.96℃ 10sec
3.55℃ 5sec
4.60℃ 3min
5.go to 2.29times
6.25℃ forever
↓
add 0.5ul PHOSPHATASE ALKALINE shrimp
↓
37℃ 1hr incubate
↓
add 1ul 3MNaOAc
↓
add 25ul EtOH
↓
20000xg 4℃ 10min centrifugation
↓
put off supernatant
↓
dry tubes
↓
add 15ul HiDi
↓
put them in the sequence machine

7/26

colony PCR again, using 7/20 protocol.
PCR program
1.95℃ 2min
2.95℃ 30sec
3.55℃ 30sec
4.72.5℃ 150sec
5.go to 2. 29times

but,unsucessful.

7/27

Miniprep of E.coli cells containing LDLR gene(that yesterday picked up and cultured) with Promega, Wizard Plus SV Miniprep DNA Purification System
using AGE, found a colony that has LDLR infusion plasmid

August

PCR of GFP for fusion
program
1.95℃ 2min
2.95℃ 30sec
3.55℃ 30sec
4.72.5℃ 20sec
5.go to 2. 29 times
6.25℃ forever

We found that the length of these PCR product till now was longer than that of LDLR.

• read the sequence of LDLR cDNA→the result didn't conform with NCBI datebase
  
• change PCR program→didn't get the PCR product whose length was correct
  

September

We got new LDLR cDNA!

• look for the best PCR program
  

→DMSO 5% or 10%
→annealing temparature 55℃ or 60℃

result
the best program
10% DMSO
1.95℃ 2min
2.95℃ 30sec
3.60℃ 30sec
4.72.5℃ 1min
5.go to 2. 29 times
6.25℃ forever
purify the PCR product using Promega kit and insert it in iGEM part(plate1-7D)

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