Report: Transformation of

E.coli

Names: Dai Mo(writer), Ye Yun, Tong Xin

Date: …Nov. 22, 2010………..

1. Religation of the original plasmid is normally prohibited by a previous

incubation with alkaline phosphatase. What does this enzyme do?

Alkaline phosphatase is a hydrolase enzyme. It can cause dephosphorylation by
removing phosphate groups from many types of molecules. In this case, alkaline
phosphatase can remove the phosphate group from the 5’ end of DNA, by which
the DNA is prevented from ligating (the 5’ end attaching to the 3’ end), thereby
keeping DNA molecules linear until the next step of the process for which they
are being prepared.

2. In our experiment no alkaline phosphatase is used. Why is it not

necessary in our case?

In our experiment, we want to find out the ratio of plasmid which are religated
with insert and plasmids which are religated without insert, so it is not
necessary to use alkaline phosphatase in this case.

3. Insert a table with all restriction fragments produced by a double digest

(E+H) of the phage  genome. Give information about the sticky ends

produced on each fragment and order them from large to small!!!

# Ends Coordinates Length(bp)  

1 LeftEnd-EcoRI 1-21226 21226
 2 EcoRI-HindIII 31748-36895 5148
3 EcoRI-HindIII 39169-44141 4973
4 HindIII-EcoRI 27480-31747 4268
5 EcoRI-RightEnd 44973-48502 3530
6 HindIII-HindIII 23131-25157 2027
7 EcoRI-HindIII 21227-23130 1904
8 HindIII-EcoRI 37460-39168 1709
9 EcoRI-HindIII 26105-27479 1375
10 HindIII-EcoRI 25158-26104 947
11 HindIII-EcoRI 44142-44972 831
12 HindIII-HindIII 36896-37459 564

# Cut position 5'…Site with flanks…3'

1 *21226/21230 21216 GGGCCGGTGA G^AATT_C GGCCTTTCCG
2 26104/26108 26094 ATGCTGAAAT G^AATT_C TAAGCGGAGA
3 31747/31751 31737 CGCCGGAAGT G^AATT_C AAACAGGGTT
4 39168/39172 39158 ATTCGTCAGA G^AATT_C TGGCGAATCC
5 44972/44976 44962 TGTCTGTCCT G^AATT_C ATTAGTAATA

Which fragments can be inserted in the pGEM-3Zf(+) vector?

In the pGEM-3Zf(+) vector, there is a EcoRI cutting site in the position 5, and a
HindIII cutting site in the position 56, so all the fragments can be inserted in the
vector.

4. Why is the vector/insert ratio important during ligation? What could

happen if this ratio is totally in favour for the vector (much more vector than

insert molecules) or on the other hand in favour for the insert (much more

insert than vector)?

The vector/insert ratio during ligation indicates how easy the insert can be
inserted into the vector and it may influence the amount of colonies afterwards.
When this ratio is in favour for the vector, there are more vectors available, so they
may ligate with themselves, and as a consequence there will be less viable colonies
produced. The same thing happens when this ratio is in favour for the inserts.

5. Make a hypothesis about the ratio: competent cells/amount of DNA, that is

present during the transformation reaction! What will happen if there are

many DNA molecules for every competent cell?

We assume that the higher the competent cells/amount of DNA ratio is, the better
the result could be. This is because if there are many DNA molecules for every
competent cell, the number of competent cells will become the limiting condition of
DNA subclone afterwards. We can not determine the amount of inserts been
inserted successfully into vectors.

6. What is the purpose of using the ‘Wizard DNA Clean-Up system (Promega

A7280) prior to ligation?

The Wizard® DNA Clean-Up System provides a simple and effective way to purify
linear and circular DNA (200–50,000bp) from many molecular biology reactions.
Using a quick batch-column procedure, the entire process can be completed in 15
minutes or less with no organic extractions or ethanol precipitations.
Using this system, we can get purified nicked DNA which is then eluted in water or
TE buffer, ready for use.

7. There exists a ‘pGEM®-T Easy Vector System I’ (Promega A1360) for the

ligation of fragments in pGEM3Zf(+). Under what circumstances can we use

this kit? (which fragments can be cloned this way?) (Use the information map

or the Promega internet site)

The pGEM®-T Easy Vector Systems are convenient systems for the cloning of PCR
products. They offer all of the advantages of the pGEM®-T Easy Vector Systems
with the added convenience of recognition sites for EcoRI and Notl flanking the
insertion site. Thus several options for removal of the desired insert DNA with a
single restriction digestion are provided.
8. Explain the blue/white screening procedure!
The pGEM-Z vector encodes the α subunit of LacZ protein with an internal multiple
cloning site (MCS), while the chromosome of the host strain encodes the remaining
Ω subunit to form a functional β-galactosidase enzyme. The MCS can be cleaved by
different restriction enzymes so that the foreign DNA can be inserted within the
lacZα gene, thus disrupting the production of functional β-galactosidase. The
chemical required for this screen is X-gal, a colourless modified galactose sugar
that is metabolized by β-galactosidase to form an insoluble product (5-bromo-4
chloroindole) which is bright blue, and thus functions as an indicator. Isopropyl β-
D-1-thiogalactopyranoside (IPTG), which functions as the inducer of the Lac
operon, can be used in some strains to enhance the phenotype. The hydrolysis of
colorless X-gal by the β-galactosidase causes the characteristic blue color in the
colonies; it shows that the colonies contain vector without insert. White colonies
indicate insertion of foreign DNA and loss of the cells' ability to hydrolyse the
marker.

9. Calculate the ratio: vector/insert during ligation, (g/g and in mol/mol)

knowing:

Conversion μg → μmol for dsDNA:

μg x (106pg/1 μg) x (pmol/660pg) x (1/N) = pmol

N = number of base pairs (MW 660/bp)

Amount pGEM-3Zf(+) used during ligation reaction: …0.005…… g/l / ……

0.0024……….. pmol/l

Size pGEM-3Zf(+) vector: ……3199………bp

 Amount  DNA used during ligation reaction: ……0.0585……… g/l /

………0.002…….. pmol/l

Size  DNA genome: …48,502……….bp

What we really want to know is the amount of phage  fragments! How

many fragments are produced using the restriction enzymes ……12…. :

So the number of phage  fragments = ………0.024…….. pmol/l

Ligation l g pmol l g pmol vector/insert vector/insert


Reaction … vector vector vector  RD  RD g/g pmol/pmol
RD
contains:
1 0.005 0.002 3 0.175 0.072 0.0285 0.033
A
4 5
3 0.015 0.007 1 0.058 0.024 0.2564 0.3
B
2 5

Fill in the table:

Ligation white blue % white Mean

mixture % white + SD
0 0 - 100+/-0
A
3 0 100
A
3 0 100
A
4 10 29 40+/-23
B
6 3 67
B
 1 3 25
B
0 204 0 0+/-0
C1
0 145 0
C2

10. What ligation mixture gives you the highest percentage transformed cells

harbouring a recombined plasmid? Mixture A (excess insert) or mixture B

(excess vector)? Explain!

Mixture A gives us the highest percentage. As mentioned in question 4, when there
are much more vectors then inserts exist, vectors may religate with themselves
instead of inserts. So there are less colonies than mixture A.

11. Calculate the transformation efficiency using the control mixture.

= number of blue colonies per amount (g) of plasmid

So you have to take into account the amount of plasmid present in a certain

culture volume (probalby 450l) and the amount of this culture that you have

applied on the petri dish!

Concentration of control plasmid: …0.01…………g/l

Ex.: C1 transformation reaction contained .…3… l control plasmid;

So there was .…0.03… g in the total end volume (450 l ) of the

transformation reaction!
 dish g l culture Number colonies colonies

plasmid in Spread on blue per 450 l per g

transf. the petri colonies (n*450/?l

reaction dish (n)

0.03 10 204 9180 306000
C1
100 273 1228.5 40950
C1
0.08 10 145 6525 81562.5
C1
100 350 1575 19687.5
C2

Mean value of transformation efficiency ………112050…….colonies/g

When comparing the competent cells for C1 and C2, we centrifuged instead of
mixing gently by mistake, this may have influenced the result.

Compare with commercial available competent cells:

Promega cat n° L1001 of L2001

Explain?
The transformation efficiency of L2001 competent cells is greater than 108cfu/μg.
Our result is much lower than this value. The reason may be that the
transformation efficiency of the commercial one is measured in ideal conditions,
while ours are not. And the competent cells/DNA ratio may also change the result.

12. Give a list of factors which might influence the transformation efficiency!
 Factors that influence transformation efficiency include technique errors,
temperature and incubation length, growth stage of cell, insert/vector ratio,
competent cells/amount of DNA ratio and etc.

13. Try to make an estimation of the ratio:

Number of bacteria/number of recombined vector molecules

During the transformation reaction

Reasoning:

A600nm of the culture: ……………

Neem aan dat er ongeveer 1.107 bacteriën/ml zijn als de A600 = 2

Voor het bereiden van getransformeerde cellen ben je vertrokken van ………

ml cultuur!

Als je aanneemt dat geen cellen verloren gaan tijdens de CaCl 2 behandeling,

dan zitten al die bacteriën uiteindelijk in ……….. CaCl2 oplossing

Tijdens de eigenlijke transformatiereactie heb je 45 l van deze behandelde

cellen toegevoegd aan het reactiemengsel; dus hierin zitten ……………….

Cellen!

Hierin zit ook …………. pmol vector (A) en …………. pmol vector (B) (zie

hierboven

1 mol bevat ……………… deeltjes; dus in A hebben ……………… moleculen

vector en in B hebben …………………moleculen vector.

Verhouding:
 - cellen/vector in A =

- cellen/vector in B =

14. What can you do to know which fragments were inserted into your

plasmid?
In order to find out which fragment were inserted into the plasmid, we need to
take out some plasmids from the white colour colony, and then digest them using
the same restriction enzyme as we used before insertion. After this, we can have
free fragments. Then we use electrophoresis to compare the fragment with 12
known fragments from phage lambda genome. By comparison, we can find out
the matched fragment and then we know which fragments were inserted.