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-Qiagen DNeasy Kit was used to extract genomic DNA from E.Coli pellets.
-PCR is performed with DinB forward (Tm: 60C) and DinB reverse (Tm: 62C) primers.
-PCR
-PCR product -
PCR product successful
-Transformation was unsuccessful
-No colonies grew on plate with what should have had DinB insert.
-Only blue colonies grew on control plate.
-Because the control came from the kit, white colonies should have grown.
-It was undetermined why there was no growth and why the control was inaccurate
-Unsuccessful control results means PCR sample needed to be re-ligated and re-transformed.
-DinB Inserted Plate: 2 white, 5 blue
-Control Plate: 4 white, 3 blue
-The next steps are to determine whether or not the white colonies that grew are our inserted f13s
-Resuspended a white colony from pGEM + DinB plate in order to
run a PCR. Running this PCR would tell if colonies were of desired
product (pGEM with the DinB insert)
-PCR is performed with SP6 (Tm: 51.5C) and T7 (Tm: 56.3C) primers.
-PCR
Primers SP6 and T7 are used because they will cleave the insert out of the vector
20 cycles:
-Denaturation: 95C for 5:30 min; 30 sec for subsequent
-Annealing: 53C for 1 min
-Extension: 72C for 1 min
-Final Extension: 72C for 10 min
-DinB insertion was successful
-Cell cultures made from two white colonies
-Plasmid DNA purified with Qiagen DNeasy Kit
-EcoRI and BamHI used
-Digestion Failed; no enzyme cut -
-Digestion Failed; concluded that purified DNA was not
completely clean, under which conditions the enzymes would
not activate -
-Mini-dialysis to clean DNA
-Re-digestion (again with EcoRI and BamHI)
**Noticed: Cells in culture were not healthy. Something about the growth environment was toxic and making calls sick.
-PCR shows successful insertion so there should should be digestion
-To investigate why the cells were unhealthy, each colony was inoculated with and without ampicillin; expected to see normal growth on plates without ampicillin and no growth on plates with amp because vectors with insert were being selected against.
-All plates grew as lawns and no individual colonies were distinguishable.
-Re-inoculated; expected to see normal growth on plates without ampicillin and no growth on plates with amp because vectors with insert were being selected against.
-Results as expected. Healthy and unheathy colonies seen on all plates.
-A healthy and an unhealthy colony were picked to be re-inoculated, respectively; expecting to see some healthy and some unhealthy colonies on the same plate. Healthy (white round colonies) are mutated to survive the toxicity of the environment. Unhealthy colonies are transparent and flat
-Results were as expected; most colonies were unhealthy
-More cells grew on plates without ampicillin than on plates with
-f13s inserted
-Healthy cells: mutated cells
-Unhealthy cells
Could be any combination of vector/insert of just vector.
-Whatever caused the cells to become sick remains undetermined. We suspect that the inserted f13s mutation was causing the colonies to be sick.
-Test both healthy and unhealthy colonies to see if they have insert. Make culture of inserted and not inserted. Observe culture to see if the cells with f13s insert get sick and the cells without it remain healthy. This will prove that the f13s is the cause of toxicity.
- Obtained low-copy plasmid DNA pBAD33 from E.Coli cells with QIAprep Miniprep Kit
- PCR was performed with our own designed forward primer and reverse primers
* Designed for EcoRI and BamHI digest
- MJ forward primer- Tm: 72.9C
5’-GGACGCGAGTACCGCTATCATGAAT
TCATGCGTAAAATGATTCATGTGGA-3’
-MJ reverse primer- Tm: 74.7C
5’-GGATCCTAATCCCAGCACCAGTTGG
TGGTGATGGTGATGATGTCA-3’
-DNA resource: F13S TLS deficient cell suspension
20 cycles:
- Denaturation: 95C for 5 min; 30 sec for subsequent
- Annealing: 55C for first three cycles (specific Tm)
67C for the rest cycles (Tm)
- Extension: 72C for 1 min
72C for 10 min for the last cycle
* Gel picture was lost, but we did get bands for PCR products
* It was also confirmed on NanoDrop; the NanoDrop gave us the PCR products had DNA concentration at ~28ng/uL
-used QIAquick PCR purification kit to purify PCR product
-F13S PCR product was ligased into pBAD33 vector which has been cut by EcoRI and BamHI
-Transferred only ligation-1 and ligation-2 tubes
-Transformed cells were plated on Chloramphenicol plates for selecting successful gene insertion.
-8 colonies out of 157 white colonies from the plate were chosen and made suspension for each colony
- Added phenol chloroform (break cells, destroy proteins, expose all DNA and RNA)
-Extracted DNA phase and ran gel electrophoresis
8 selected colonies were re-inoculated for future use
gel from dirty prep colonies
1~8 & uncut pBAD33
-ran PCR with DinB forward and DinB reverse primers
-gel eletrophoresis
- Dirty prep didn't show obvious evidence about insertion
- PCR with DinBF and DinBR showed a band, but this band didn't show correct size (1kb)
- Double digest didn't cut the plasmid into right sizes; rather, it showed "gross re-arrangement"
Concluded that cells from colony 1 there was no successful insertion.
Results from dirty prep were not very reliable and obvious. We may want to inoculate more colony suspension for PCR and double digest test. We may see some colonies have successful insertion, while some don't (or may be gross-arranged).
-5 tubes prpared with LB/Ampicillin solution
-Tube 1 is introduced with 100µL of IPTG
solution is diluted down each tube by 100µL
-Samples are prepared and for sodium dodecyl sulfate polyacrylamide gel electrophoresis.
-Gel Results
When LB/DinBC66A-CHis cell culture was at
OD 0.711, ITPG was added at 1.25mmol
-Cells were harvested and proteins extracted according to the Novagen Bugbuster Protein Extraction Reagent Kit
-SDS determines the size of the proteins
-Protein should be seen in column 3 and 6
- We expect our protein size to be ~30-35 kDa
-Wash 1 should show a lot of debris, but no protein in colums 4 and 6
-Last wash sould show nothing in columns 5 and 8
-The load should show everything including protein column 9
-Gel Results
-The IPTG was successful in eliminating the lac repression and it successfully expressed the proteins.
-DNA recombination technology for
Protein Overexpression
- Ran extracted pBAD 33 on gel electrophoresis
* Lost the gel picture
Cells are resuspended and a culture
is grown
pGEM PCR Product
Gel run on 2.17 with
2.10 PCR
Induces lac operon expression in E. coli by binding to the lacI
repressor, preventing repression of lacZ.
PCR
Product
20 cycles:
-Denaturation: 95C for 1 min; 30 sec for subsequent
-Annealing: 55C for 1 min
-Extension: 72C for 1 min
-Final extension: 72C for 10 min
Painful memory of numerous unsuccessful digests --Yeah~! Finally!
BamHI
BamHI & EcoRI double digest
uncut
BamHI
EcoRI
EcoRI & BamHI
double digest
EcoRI
2.10
Gel for EcoRI and BamHI
double digest (1)
Gel for EcoRI and BamHI
double digest (2)
2.24
3.7
Gel for EcoRI and BamHI
double digest
-Promega ligation protocol followed
-f13s PCR product from 2/17 ligated into the pGEM and the control from kit is used
Lane 1 & 8: Ladder
Lane 2-7: Highest to lowest IPTG concentration
Lane 4 (second dilution) used as concentraion for culture
-Magnetic agarose beads are used to bind the proteins while all cellular debris and dna is washed away (4 washes)
Well layout
1) BIO-RAD precision plus protein unstained standard--Ladder
2) Skip (ladder spilt out)
3) Protein elution 1
4) First wash of Protein elution 1
5) Last wash of Protein elution 1
6) Protein elution 2
7) First wash of Protein elution 2
8) Last wash of Protein elution 2
9) Load
-PCR setting:
Gel run on 4.4
Dyed and imaged on 4.5
-PCR product
pGEM PCR Product
Gel run on 3.10
DinB insert
-PCR Product-
Double Digestion
Gel run on 3.14
-Re-digested (again with EcoRI and BamHI)
Double Digestion
Gel run on 3.17
-Digestion Failed -
Double Digestion
2036 bp
1636 bp
Gel run on 3.21 with
3.17 digest
150 ng : 50 ng
50 ng : 16 ng
1:3 (pBAD33:PCR product)
3:1 (pBAD33:PCR product)
no PCR product--control
* pBAD33: PCR product is the ratio in nano-gram
Colonies plated on 3.21
Growth observed on 3.24
Imaged on 4.7
Healthy
Unhealthy
Ampicillin No Ampicillin
what we expect from
dirty-prep gel
-Different types of cells
As seen in colony growth
heavy dye
gel from dirty prep colonies
1~8 & uncut pBAD33
***dye washed***
PCR of ligated pBAD33
Gel of double
digested pBAD33
4/4/11